Methods of making hemagglutinin proteins

ABSTRACT

Methods of making a protein that stimulates a protective immune response in a subject include separating a portion of a protein from a naturally occurring influenza viral hemagglutinin to form a protein portion. The protein portion includes at least a portion of a globular head, and at least a portion of at least one secondary structure having at least one β-sheet at a bottom of the globular head that causes the globular head to essentially retain its tertiary structure. The protein portion made by the methods of the invention lacks a transmembrane domain, a cytoplasmic domain and an HA2 subunit. A nucleic acid sequence encoding the protein portion is transformed into a prokaryotic host cell.

RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.11/714,873, filed on Mar. 6, 2007, which claims the benefit of U.S.Provisional Application Nos. 60/779,854, filed on Mar. 7, 2006;60/784,497, filed on Mar. 20, 2006; 60/790,457, filed on Apr. 7, 2006;60/814,292, filed on Jun. 16, 2006; 60/830,881, filed on Jul. 14, 2006;60/838,007, filed on Aug. 16, 2006; and 60/856,451, filed on Nov. 3,2006. The entire teachings of the above applications are incorporatedherein by reference.

INCORPORATION BY REFERENCE OF MATERIAL IN ASCII TEXT FILE

This application incorporates by reference the Sequence Listingcontained in the following ASCII text file being submitted concurrentlyherewith:

File name: 37101021020SubSequenceListing.txt; created Mar. 12, 2013, 988KB in size.

BACKGROUND OF THE INVENTION

Viral influenza infection can lead to disease. Strategies to prevent andmanage disease associated with viral influenza infection can includevaccines with inactivated viruses and drugs. However, such strategiescan be costly to maintain supply with demand and, thus, be limited insupply; may result in variable protection and less than satisfactoryalleviation of symptoms, thereby ineffectively preventing or treatingillness and, in some instances death, consequent to disease associatedwith viral influenza infection. Thus, there is a need to develop new,improved and effective methods of treatment for preventing and managingdisease associated with viral influenza infection.

SUMMARY OF THE INVENTION

The present invention relates to compositions, such as compositions thatstimulate a protective immune response, and methods of making proteinsthat stimulate a protective immune response in a subject.

In one embodiment, the invention is a method of making a protein thatstimulates a protective immune response in a subject, comprising thesteps of separating a portion of a protein from a naturally occurringviral hemagglutinin to thereby form a protein portion, wherein theprotein portion includes at least a portion of a globular head, and atleast a portion of at least one secondary structure that causes theglobular head to essentially retain its tertiary structure, and whereinthe protein portion lacks a membrane fusion domain, a transmembranedomain and a cytoplasmic domain; transforming a nucleic acid sequenceencoding the protein portion into a prokaryotic host cell; and culturingthe prokaryotic host cell to thereby make the protein that stimulates aprotective immune response in a subject.

In another embodiment, the invention is a method of making a proteinthat stimulates a protective immune response in a subject, comprisingthe steps of separating a portion of a protein from a naturallyoccurring viral hemagglutinin to thereby form a protein portion, whereinthe protein portion includes at least a portion of a globular head, andat least a portion of at least one secondary structure that causes theglobular head to essentially retain its tertiary structure, and whereinthe protein portion lacks a membrane fusion domain, a transmembranedomain and a cytoplasmic domain; transfecting a nucleic acid sequenceencoding the protein portion into a eukaryotic host cell, wherein theeukaryotic host cell is not a Pichia pastoris eukaryotic host cell; andculturing the eukaryotic host cell to thereby make the protein thatstimulates a protective immune response in a subject.

In another embodiment, the invention is a method of making a proteinthat stimulates a protective immune response in a subject, comprisingthe steps of separating a portion of a protein from a naturallyoccurring viral hemagglutinin to thereby form a protein portion, whereinthe protein portion includes at least a portion of a globular head, andat least a portion of at least one secondary structure that causes theglobular head to essentially retain its tertiary structure, and whereinthe protein portion lacks a membrane fusion domain, a transmembranedomain and a cytoplasmic domain; transfecting a nucleic acid sequenceencoding the protein portion into a eukaryotic host cell, wherein theeukaryotic host cell is not a Drosophila melanogaster eukaryotic hostcell; and culturing the eukaryotic host cell to thereby make the proteinthat stimulates a protective immune response in a subject.

In a further embodiment, the invention is a method of making a proteinthat stimulates a protective immune response in a subject, comprisingthe steps of separating a portion of a protein from a naturallyoccurring viral hemagglutinin to thereby form a protein portion, whereinthe protein portion includes at least a portion of a globular head, andat least a portion of at least one secondary structure that causes theglobular head to essentially retain its tertiary structure, and whereinthe protein portion lacks a membrane fusion domain, a transmembranedomain and a cytoplasmic domain; transfecting a nucleic acid sequenceencoding the protein portion into a eukaryotic host cell, wherein theeukaryotic host cell is not an insect eukaryotic host cell; andculturing the eukaryotic host cell to thereby make the protein thatstimulates a protective immune response in a subject.

In a further embodiment, the invention is a method of making a proteinthat stimulates a protective immune response in a subject, comprisingthe steps of separating a portion of a protein from a naturallyoccurring viral hemagglutinin to thereby form a protein portion, whereinthe protein portion includes at least a portion of a globular head, andat least a portion of at least one secondary structure that causes theglobular head to essentially retain its tertiary structure, and whereinthe protein portion lacks a membrane fusion domain, a transmembranedomain and a cytoplasmic domain; transfecting a nucleic acid sequenceencoding the protein portion into a eukaryotic host cell, wherein theeukaryotic host cell is not a stably transformed insect host cell; andculturing the eukaryotic host cell to thereby make the protein thatstimulates a protective immune response in a subject.

In still another embodiment, the invention is a method of making aprotein that stimulates a protective immune response in a subject,comprising the steps of separating a portion of a protein from anaturally occurring viral hemagglutinin to thereby form a proteinportion, wherein the protein portion includes at least a portion of aglobular head, and at least a portion of at least one secondarystructure that causes the globular head to essentially retain itstertiary structure, and wherein the protein portion lacks a membranefusion domain, a transmembrane domain and a cytoplasmic domain;transfecting a nucleic acid sequence encoding the protein portion into aeukaryotic host cell, wherein the eukaryotic host cell is neither aPichia pastoris eukaryotic host cell nor a stably transfected insecthost cell; and culturing the eukaryotic host cell to thereby make theprotein that stimulates a protective immune response in a subject.

In yet another embodiment, the invention is a method of stimulatingprotective immunity in a subject, comprising the step of administeringto the subject a composition that includes a protein made by a methodcomprising the steps of separating a portion of a protein from anaturally occurring viral hemagglutinin to thereby form a proteinportion, wherein the protein portion includes at least a portion of aglobular head, and at least a portion of at least one secondarystructure that causes the globular head to essentially retain itstertiary structure, and wherein the protein portion lacks a membranefusion domain, a transmembrane domain and a cytoplasmic domain;transforming a nucleic acid sequence encoding the portion into aprokaryotic host cell; and culturing the prokaryotic host cell tothereby make the protein that stimulates a protective immune response ina subject.

In an additional embodiment, the invention is a method of stimulatingprotective immunity in a subject, comprising the step of administeringto the subject a composition that includes a protein portion of anaturally occurring viral hemagglutinin, wherein the protein portionincludes at least a portion of a globular head, and at least a portionof one secondary structure that causes the globular head to essentiallyretain its tertiary structure and wherein the protein portion lacks amembrane fusion domain, a transmembrane domain and a cytoplasmic domain.

In still another embodiment, the invention is a method of making a viralhemagglutinin protein that stimulates a protective immune response in asubject, comprising the steps of separating a portion of a protein froma naturally occurring viral hemagglutinin to thereby form a proteinportion, wherein the protein portion includes at least a portion of aglobular head and at least a portion of at least one secondary structurethat causes the globular head to essentially retain its tertiarystructure, and wherein the protein portion lacks a membrane fusiondomain, a transmembrane domain and a cytoplasmic domain; transfecting anucleic acid sequence encoding the portion in a eukaryotic host cell,wherein the eukaryotic host cell is neither a Pichia pastoris eukaryotichost cell nor a stably transfected insect host cell; and culturing theeukaryotic host cell to thereby make the protein that stimulates aprotective immune response in a subject.

In another embodiment, the invention is a method of making a proteinthat stimulates a protective immune response in a subject, comprisingthe steps of separating a portion of a protein from a naturallyoccurring viral hemagglutinin to thereby make a protein portion, whereinthe protein portion includes at least a portion of a globular head, andat least a portion of at least one secondary structure that causes theglobular head to essentially retain its tertiary structure, and whereinthe protein portion lacks a membrane fusion domain, a transmembranedomain and a cytoplasmic domain; infecting a nucleic acid sequenceencoding the protein portion into an insect cell host cell; andculturing the insect host cell to thereby make the protein thatstimulates a protective immune response in a subject.

In another embodiment, the invention is a method of making a viralhemagglutinin protein that stimulates a protective immune response in asubject, comprising the steps of transforming a prokaryotic host cellwith a nucleic acid sequence encoding at least one viral hemagglutininthat lacks a transmembrane domain and a cytoplasmic domain; andculturing the prokaryotic cell to thereby make the protein.

In a further embodiment, the invention is a method of stimulatingprotective immunity in a subject, comprising the step of administeringto the subject a composition that includes a protein made by a methodcomprising the steps of transforming a prokaryotic host cell with anucleic acid sequence encoding at least one viral hemagglutinin thatlacks a transmembrane domain and a cytoplasmic domain; and culturing theprokaryotic host cell to thereby make the protein.

In another embodiment, the invention is a method of stimulatingprotective immunity in a subject, comprising the step of administeringto the subject a composition that includes a protein having at least oneviral hemagglutinin that lacks a transmembrane domain and a cytoplasmicdomain, wherein the protein was expressed in a prokaryotic cell.

In an additional embodiment, the invention is a composition comprisingat least a portion of at least one pathogen-associated molecular patternand a portion of a protein of a naturally occurring viral hemagglutinin,wherein the portion of the naturally occurring viral hemagglutininincludes at least a portion of a globular head and at least a portion ofat least one secondary structure that causes the globular head toessentially retain its tertiary structure, and wherein the portion ofthe naturally occurring viral hemagglutinin lacks a membrane fusiondomain, a transmembrane domain, and a cytoplasmic domain.

Another embodiment of the invention is a composition comprising aflagellin component that is at least a portion of a flagellin, whereinthe flagellin component includes at least one cysteine residue andwhereby the flagellin component activates a Toll-like Receptor 5.

In still another embodiment, the invention is a composition comprising aToll-like Receptor agonist component that is at least a portion of aToll-like Receptor agonist, wherein the Toll-like Receptor agonistcomponent includes at least one cysteine residue in a position where acysteine residue does not occur in the native Toll-like Receptoragonist, whereby the Toll-like Receptor agonist component activates aToll-like Receptor.

In an additional embodiment, the invention is a composition comprising aflagellin component that is at least a portion of a flagellin, whereinat least one lysine of the flagellin component has been substituted withat least one arginine, whereby the flagellin component activates aToll-like Receptor 5.

In yet another embodiment, the invention is a composition comprising aflagellin component that is at least a portion of a flagellin, whereinat least one lysine of the flagellin component has been substituted withat least one serine residue, whereby the flagellin component activates aToll-like Receptor 5.

Another embodiment of the invention is a composition comprising aflagellin component that is at least a portion of a flagellin, whereinat least one lysine of the flagellin component has been substituted withat least one histidine residue, whereby the flagellin componentactivates a Toll-like Receptor 5.

A further embodiment of the invention is a method of stimulating animmune response in a subject, comprising the step of administering tothe subject a composition that includes a flagellin component that is atleast a portion of a flagellin, wherein the flagellin component includesat least one cysteine residue and whereby the flagellin componentactivates a Toll-like Receptor 5.

In still another embodiment, the invention is a method of stimulating animmune response in a subject, comprising the step of administering tothe subject a composition that includes a flagellin component that is atleast a portion of a flagellin, wherein at least one lysine of theflagellin component has been substituted with at least one arginine,whereby the flagellin component activates a Toll-like Receptor 5.

An additional embodiment of the invention is a method of stimulating animmune response in a subject, comprising the step of administering tothe subject a composition that includes a flagellin component that is atleast a portion of a flagellin, wherein at least one lysine of theflagellin component has been substituted with at least one serineresidue, whereby the flagellin component activates a Toll-like Receptor5.

In another embodiment, the invention is a method of stimulating animmune response in a subject, comprising the step of administering tothe subject a composition that includes a flagellin component that is atleast a portion of a flagellin, wherein at least one lysine of theflagellin component has been substituted with at least one histidineresidue, whereby the flagellin component activates a Toll-like Receptor5.

In yet another embodiment, the invention is a method of stimulating animmune response in a subject, comprising the step of administering tothe subject a composition that includes a Toll-like Receptor agonistcomponent that is at least a portion of a Toll-like Receptor agonist,wherein the Toll-like Receptor agonist component includes at least onecysteine residue in a position where a cysteine residue does not occurin the native Toll-like Receptor agonist, whereby the Toll-like Receptoragonist component activates a Toll-like Receptor agonist.

The methods and composition of the invention can be employed tostimulate an immune response, in particular, a protective immuneresponse, in a subject. Advantages of the claimed invention include, forexample, cost effective methods and compositions that can be produced inrelatively large quantities for use in the prevention and treatment ofdisease associated with viral influenza infection. The claimed methodsand compositions can be employed to prevent or treat viral influenzainfection and, therefore, avoid serious illness and death consequent toviral influenza infection.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts a ribbon diagram of PR8 influenza A hemagglutin (HA)crystal structure (1RU7). Schematically presentations from left to rightare HA trimer, monomer, the globular head domain and the three choicesof vaccine candidates. Molecules or domains of interest are highlightedby dotted circle, such as the monomer in trimer structure and theglobular head domain within the monomer structure. The domain boundariesof three vaccine candidates are marked by the cross signs. The residuenumbers of each selection are also labeled in the individual vaccinepresentation.

FIG. 2 depicts a hydrophobicity plot analysis of the B/Lee/40 HA (SEQ IDNO: 36) using ProtScale (http://ca.expasy.org/tools→Primary structureanalysis→ProtScale) to confirm that the selected boundaries were in thehydrophilic regions of the protein.

FIG. 3 depicts a sandwich ELISA analysis of STF2.HA B strain proteins.ELISA plates were coated with antibody to flagellin, and the indicatedproteins were incubated to allow capture. Proteins were detected usingferret antisera against B/Malaysia/2506/2004 and enzyme-labeled goatanti-ferret antibodies.

FIG. 4 depicts the TLR bioactivity of STF2.HA B strain proteins. HEK293(TLR5+) cells were incubated with the indicated proteins overnight, andthe cell culture supernatants were harvested and assessed for IL-8 byELISA.

FIG. 5 depicts the TLR bioactivity of STF2.HA B strain proteins. HEK293(TLR5+) cells were incubated with the indicated proteins overnight, andthe cell culture supernatants were harvested and assessed for IL-8 byELISA.

FIG. 6 depicts the dose-dependent antibody response to STF2.HA1-2(PR8)(SEQ ID NO: 90) in BALB/c mice. Mice (10/group) were immunized asindicated on days 0 and 14, and bled on days 12 and 21. Anti-HA IgGresponses on Day 28 were examined by ELISA. Convalescent antisera (x)were included as a positive control. The data depict the mean±SD of 10individual sera per group.

FIG. 7 depicts serum reactivity with influenza infected cells in vitro.The day 21 serum samples depicted in FIG. 6 were incubated with mock-and PR/8/34-infected MDCK cells. The data represent the OD₄₅₀ mean±SD of10 individual sera/group.

FIG. 8A depicts survival of BALB/c mice immunized with STF2.HA1-2(PR8)(SEQ ID NO: 90). Mice from FIGS. 8 and 9 were challenged on day 28 withan LD₉₀ (8×10³ EID) of influenza A PR/8/34 administered intranasally(Day 0 post-challenge).

FIG. 8B depicts weights of BALB/c mice immunized with STF2.HA1-2(PR8)(SEQ ID NO: 90). Mice from FIGS. 6 and 7 were challenged on day 28 withan LD₉₀ (8×10³ EID) of influenza A PR/8/34 administered intranasally(Day 0 post-challenge). Graph reflects the average weight per groupbased on individual animals measured daily for 19 days.

FIG. 8C depicts clinical Score of BALB/c mice immunized withSTF2.HA1-2(PR8) (SEQ ID: NO 90). Mice from FIGS. 6 and 7 were challengedon day 28 with an LD₉₀ (8×10³ EID) of influenza A PR/8/34 administeredintranasally (Day 0 post-challenge). Graph reflects the average clinicalscore per group based on individual animals measured daily for 19 days.Clinical scores were assessed as follows: 4 pts=healthy, 3 pts=reducedgrooming, 2 pts=reduced physical activity and 1 pt=moribund.

FIG. 9 depicts neutralization activity of immune sera from BALB/c miceimmunized with the indicated doses of STF2.HA1-2(PR8) (SEQ ID NO: 90) ondays 0 and 14, and bled on day 21. The endpoint titer of each sample wascalculated as the titer that inhibited virus-mediated lysis of the cellsby at least 50%. Geometric mean titers±SD were calculated for each dosegroup and plotted.

FIGS. 10A and 10B depict anti-HA antibody response of BALB/c miceimmunized with 3 μg of STF2.HA1-2(PR8) (SEQ ID NO: 90), or 3 or 0.3 μgof STF2.HA1-2(VN) (SEQ ID NO: 95) s.c. on days 0 and 14. On day 21, serawere isolated and examined for reactivity with (FIG. 10A) HA purifiedfrom Influenza A/Viet Nam/1203/2004 (obtained from BEI ResourcesCat#NR-660)) and (FIG. 10B) recombinant flagellin (STF2) (SEQ ID NO: 96)by ELISA.

FIG. 11 depicts a flow chart of the steps required to express pFastBacconstructs using Bac-to-Bac Baculovirus Expression system.

FIGS. 12A and 12B depict an anti-HA specific IgG responses in BALB/cmice immunized with STF2.HA1-1 fusion proteins. Mice (10/group) wereimmunized with indicated STF2.HA1-1 fusion proteins on days 0 and 14,and bled on day 21. Anti-HA and anti-flagellin IgG responses wereexamined by ELISA. Convalescent antisera were included as a positivecontrol. The data depict the mean±SD of 10 individual sera per group.

FIG. 13A depicts survival of BALB/c mice immunized with recombinantSTF2.HA1-1 proteins. On day 28, animals in FIG. 12 were challenged i.n.with an LD₉₀ (8×10³ EID) of influenza A PR/8/34. The survival ofindividual mice was monitored for 21 days post challenge.

FIG. 13B depicts weight of BALB/c mice immunized with recombinantSTF2.HA1-1 proteins. On day 28, animals in FIG. 12 were challenged i.n.with an LD₉₀ (8×10³ EID) of influenza A PR/8/34. Graph reflects theaverage weight per group based on individual animals measured daily for21 days.

FIG. 13C depicts clinical assessment of BALB/c mice immunized withrecombinant STF2.HA1-1 proteins. On day 28, animals in FIG. 12 werechallenged intranasally (i.n.) with an LD₉₀ (8×10³ EID) of influenza APR/8/34. Graph reflects the average weight per group based on individualanimals measured daily for 21 days. Clinical scores were assessed asfollows: 4 pts=healthy, 3 pts=reduced grooming, 2 pts=reduced physicalactivity and 1 pt=moribund.

FIG. 14 depicts anti-HA antibody titers of mice following immunizationwith 10 μg STF2.HA1-2(VN).

FIG. 15 depicts an anti-HA antibody titers of mice followingimmunization with 3 μg STF2.HA1-2(VN).

FIG. 16 depicts an anti-HA antibody titers of mice followingimmunization with 1 μg STF2.HA1-2(VN).

FIG. 17 depicts a survival of mice following challenge with 10 LD₉₀ ofinfluenza A/Viet Nam/1203/04

FIG. 18 depicts the amino acid sequence of full-length flagellin (STF2)(SEQ ID NO 312) highlighting important domains and residues.Gray=experimentally-defined TLR5 binding site (Smith, et al., 2003);K=lysine residues; underline=flagellin sequences corresponding to STF2Δ,construct (linker not shown).

FIG. 19 depicts a space-filling model of flagellin (STF2) with the TLR5activation site (left panel) and lysine residues (right panel)highlighted in gray.

FIG. 20 depicts a space-filling model of flagellin (STF2) with residuesretained in STF2Δ highlighted in black.

FIG. 21 depicts a schematic depiction of the two-step PCR cloning ofpMT/STF2Δ.

FIG. 22 depicts recognition of flagellin epitopes of STFΔ proteins inELISA.

FIG. 23 depicts TLR5-dependent secretion of IL-8 following stimulationof HEK293 cells with the indicated STF2Δ proteins.

FIG. 24 depicts the 5200 chromatogram of STF2Δ.HingeCys:CysH1C1.Void=column void volume; conjugate=elution peak of protein: peptideconjugate; A260, A280 and conductivity are indicated.

FIG. 25 depicts TLR5-dependent secretion of IL-8 following stimulationof RAWhTLR5 cells (closed symbols) or TLR5-negative RAW cells (opensymbols) with STF2Δ.HingeCys (squares), STF2Δ.HingeCys:CysH1C1 conjugate(circles), or STF2.OVA (triangles).

FIG. 26 depicts the S200 chromatogram of STF2.4×H1C1. Void=column voidvolume; conjugate=elution peak of protein: peptide conjugate; A260, A280and conductivity are indicated.

FIG. 27 depicts an anti-H1C1 antibody response of BALB/c mice immunizedwith native H1C1 peptide or Pam3Cys.H1C1 peptide.

FIG. 28 depicts a survival plot of BALB/c mice immunized with nativeH1C1 peptide or Pam3Cys.H1C1 peptide then challenged with an LD₉₀ ofinfluenza A/Puerto Rico/8/34 virus.

FIG. 29 depicts the amino acid sequence of Salmonella typhimuriumflagellin type 2 (fljB/STF2) with the hinge region underlined (SEQ IDNO: 498).

FIG. 30 depicts the nucleic acid sequence (SEQ ID NO: 499) encoding SEQID NO: 498. The nucleic acid sequence encoding the hinge region isunderlined.

FIG. 31 depicts the amino acid sequence of fljB/STF2 without the hingeregion (also referred to herein as “fljB/STF2Δ” or “STF2Δ”) (SEQ ID NO:500).

FIG. 32 depicts the nucleic acid sequence (SEQ ID NO: 501) encoding SEQID NO: 500.

FIG. 33 depicts the amino acid sequence of E. coli flagellin fliC (alsoreferred to herein as “E. coli fliC”) with the hinge region underlined(SEQ ID NO: 502).

FIG. 34 depicts the nucleic acid sequence (SEQ ID NO: 503) encoding SEQID NO: 502. The nucleic acid sequence encoding the hinge region isunderlined.

FIG. 35 depicts the amino acid sequence of S. muenchen flagellin fliC(also referred to herein as “S. muenchen fliC”) with the hinge regionunderlined (SEQ ID NO: 504).

FIG. 36 depicts the nucleic acid sequence (SEQ ID NO: 505) encoding SEQID NO:504. The nucleic acid sequence encoding the hinge region isunderlined.

FIG. 37 depicts the amino acid sequence of pMT/STF2. The linker isunderlined and the sequence of the BiP secretion signal is bolded (SEQID NO: 506).

FIG. 38 depicts the nucleic acid sequence (SEQ ID NO: 507) of SEQ ID NO:506. The nucleic acid sequence encoding the linker is underlined and thenucleic acid sequence encoding the BiP sequence is bolded.

FIG. 39 depicts a Pam3Cys.M2e fusion protein. The amino acid sequence(SEQ ID NO: 510) of M2e is shown in bold type.

FIG. 40 depicts the activation of an antigen-presenting cell (APC) byToll-like Receptor (TLR) signaling.

FIGS. 41A and 41B depict plasmid constructs to express an amino-terminusof an M2 (e.g., SEQ ID NOS: 510, 554) of H1 and H5 (SEQ ID NO: 536)influenza A viral isolates. pMT: metallothionein promoter-basedexpression vector. BiP: secretion signal sequence ofimmunoglobulin-binding protein. STF2: full-length flagellin of S.typhimurium. STF2Δ: hinge region-deleted STF2. MCS: multiple cloningsite.

FIG. 42 depicts plasmid constructs designed to express HA of H1 and H5influenza A virus isolates. AOX1: AOX1 promoter of pPICZα expressionvector (Invitrogen Corporation, Carlsbad, Calif.). αf: secretion signalsequence of yeast. STF2: full-length flagellin of S. typhimurium. STF2Δ:hinge region-deleted STF2. MCS: multiple cloning site.

FIG. 43 depicts the amino acid sequence (SEQ ID NO: 561) of HA (PR8).

FIG. 44 depicts the nucleic acid sequence (SEQ ID NO: 562) encoding SEQID NO: 561.

FIG. 45 depicts the amino acid sequence (SEQ ID NO: 563) of E. coli fliCwithout the hinge region.

FIG. 46 depicts the amino acid sequence of pMT/STF2Δ. (SEQ ID NO: 585).The linker sequence is underlined and the BiP secretion signal isbolded.

FIG. 47 depicts the nucleic acid sequence (SEQ ID NO: 586) encoding SEQID NO: 585. The nucleic acid sequence encoding the linker is underlinedand the nucleic acid sequence encoding the BiP secretion signal isbolded.

FIG. 48 depicts the amino acid sequence (SEQ ID NO: 595) of theSalmonella muenchen fliC without the hinge region, which is alsoreferred to herein as “S. muenchen fliCΔ.”

FIG. 49 depicts the nucleic acid sequence of Salmonella muenchen fliC(SEQ ID NO: 596) encoding SEQ ID NO: 595.

FIG. 50 depicts IL-8 secretion following stimulation of TLR5+ cells.

FIG. 51 depicts TNF secretion following stimulation of TLR2+ cells.

FIG. 52 depicts M2e-specific IgG.

FIG. 53 depicts the OVA-specific IgG.

FIG. 54 depicts the M2e-specific IgG serum titers.

FIG. 55 depicts the M2e-specific serum IgG titer post-boost.

FIG. 56 depicts the Pam3Cys.M2e dose response.

FIG. 57 depicts the M2e-specific serum IgG titer.

FIG. 58 depicts the rabbit IgG response to M2e.

FIG. 59 depicts the immunogenicity of STF2.4×M2e in a rabbit 14 dayspost-prime.

FIG. 60 depicts the survival of BALB1c mice following challenge. Micewere immunized on days 0 and 14 as indicated in the legend andchallenged on day 28 by intranasal administration of an LD₉₀ ofinfluenza A/Puerto Rico/8/34 virus (Day 0 post-challenge). Mice weremonitored for survival for 21 days post-challenge.

FIG. 61 depicts fusion constructs in a pET24 vector. T7:T7 promoter;lacO: lac operator; STF2: Salmonella typhimurium flagellin; STF2Δ=STF2with the hinge region deleted; EIII⁺ is domain III of a West Nileenvelope protein with 6 amino acids of domain I amino acid.

FIGS. 62A and 62B depict TLR5 bioactivity of STF2.EIII+ (SEQ ID NOS:657, 658) and STF2ΔEIII+ (SEQ ID NOS: 673, 674) fusion proteins. Serialdilutions of purified proteins were added to HEK293 (TLR5+) cellsovernight and IL-8 content of the supernatants measured by ELISA.Purified STF2.OVA was used as a positive control (FIG. 72A). The TLR2agonist Pam3CSK4 was used as a negative control (FIG. 72B).

FIG. 63 depicts STF2Δ.EIII+ antigenic epitopes assessed by ELISA. Plateswere coated with full-length WNE (open bars) (SEQ ID NO: 642) orSTF2Δ.EIII+ (SEQ ID NOS: 673,674) and probed with the indicatedantibodies (mAb). Poly=polyclonal antiserum to WNE; 3D9 through7H2=neutralizing monoclonal antibodies to WNE epitopes;anti-flagellin=monoclonal antibody to flagellin.

FIGS. 64A, 64B, 64C and 64D depict reactivity of STF2.E (SEQ ID NOS:761, 762); STF2.EIII+ (SEQ ID NOS:657, 658) and STF2Δ.EIII+ (SEQ IDNOS:673,674) fusion proteins with antibodies to WNE and flagellin.Plates were coated with fusion proteins, blocked and incubated withantibodies to WNE or flagellin. Antibody reactivity was detectedfollowing incubation with HRP-labeled species specific IgG. Plates weredeveloped in the presence of TMB substrate and O.D.450/650 using a TECANplate reader and Magellian software.

FIG. 65 depicts IgG serum following injection with fusion proteins. Micewere immunized with either PBS, Drosophila conditioned medium containingSTF2.E (CM, positive control), 25 μg of STF2Δ.EIII+ (SEQ ID NOS:673,674) i.p., 25 μg STF2Δ.EIII+ s.c., 25 μg STF2.EIII+ (SEQ ID NO:659,658)i.p., 25 μg STF2.EIII+ (SEQ ID NOS:657,658) or 25 μg STF2.E (SEQ ID NOS:761, 762). On day 35, immunized animals were challenged with WNV. Serafrom individual mice (day 35) were characterized by direct ELISA todetermine IgG levels. Purified WNV-E protein (SEQ ID NO:642) was used asthe antigen in this assay. This antigen (60) was produced in Drosophilaas a his-tagged protein.

FIG. 66 depicts STF2Δ.EIII+ (SEQ ID NOS: 673 674) and STF2.EIII+ (SEQ IDNOS: 657, 658) protective immunity to WNV viral challenge. Mice wereimmunized and challenged with a lethal dose of WNV strain 2741 on day35. Survival was monitored for 21 days.

FIG. 67 depicts IgG sera titers following immunization with fusionproteins. STF2Δ.EIII+ proteins induce WNV-specific IgG antibodies. Micewere immunized s.c. on days 0, 14 and 28 with PBS alone or about 25 μgof STF2Δ.EIII+ (SEQ ID NOS: 673, 674) (045 [positive control]),STF2Δ.EIII+ (067, trimer), STF2Δ.EIII+ (070, monomer) or STF2Δ.EIIIs+(SEQ ID NOS: 675, 676) (069). On day 35 sera from individual mice werecharacterized by direct ELISA to determine IgG levels. Purified WNV-Eprotein (060, produced in Drosophila as a his-tagged protein) was usedas the antigen in this assay.

FIG. 68 depicts STF2Δ.EIII+ (SEQ ID NOS: 673, 674) and STF2Δ.EIIIs+ (SEQID NOS: 675, 673) protective immunity in mice from WNV lethal challenge.On day 38 following immunization with fusion proteins, all groups werechallenged with a lethal dose of WNV strain 2741 and survival wasmonitored for 21 days. Survival for each group (10 mice/group) isindicated as a percentage.

FIG. 69 depicts competition assays. Serial dilutions (five fold startingat 1:25) of antisera from immunized animals were incubated withbiotinylated WNE protein (SEQ ID NO: 642) and then added to the wells ofELISA plates coated with mAb 7H2 at about 2 mg/ml. Wells were developedusing avidin-HRP to determine inhibition of West Nile protein binding asa results of competition with mAb 7H2.

FIG. 70 depicts epitope mapping of the antibody response induced bySTF2Δ.EIII+ (SEQ ID NOS: 675,676) fusion proteins. Immune sera fromanimals immunized with indicated STF2Δ-fusion proteins (E2-21, E27-E52)were examined for the ability to recognize overlapping peptidescorresponding to the junction of domains I and III of the WNV envelopeprotein.

FIG. 71 depicts epitope mapping of the antibody response induced bySTFΔ.EIIIs+ (SEQ ID NOS: 675, 676) E-21 (envelope protein) epitopefusion proteins. Immune sera from animals immunized with the indicatedSTF2Δ-fusion proteins (E2-21, E2-21-1(S,C), E2-21-2(C,S), E2-21-2(C,S)and E2-21-4 through E2-21-24) were evaluated to identify the residuesdefining the E-21 epitope of West Nile envelope protein. Data reflectsthe response of sera to E-21 following the substitution of cysteine withserine (indicated by C,S); and the sequential replacement of amino acidswith alanine.

FIG. 72 depicts Pam3Cys.WNV001 (SEQ ID NO: 771) inducing EIII specificIgG antibodies. Mice were immunized s.c. on days 0, 14 and 28 with PBSalone, 22 mg of unmodified WNV001 (SEQ ID NO: 771) or 30 μg ofPam3Cys.WNV001. On day 35 sera from individual mice were characterizedby direct ELISA to determine IgG levels to synthetic WNV001 peptide.

FIG. 73 depicts the amino acid sequences (SEQ ID NOS: 691-698) of theEI/EIII junction for West Nile, Japanese encephalitis and Dengue(serotypes 1 through 4) viruses. The West Nile epitope identified usingantisera from STF2Δ.EIIIs+ immunized animals is underlined. Thissequence corresponds to peptide E2-21 (SEQ ID NO: 728).

FIG. 74 depicts a tripalmitoylated peptide that includes CSSN (SEQ IDNO: 842).

FIG. 75 depicts the D1 domain, D2 domain, TLR5 activation domain andhypervariable (D3 domain) of flagellin.

FIG. 76 depicts the D1 domain, D2 domain, TLR5 activation domain andhypervariable (D3 domain) of flagellin (Yonekura, et al. Nature 424,643-650 (2003)).

FIG. 77 depicts the D0, D1, D2 and D3 domains of flagellin and regionsin the D2 and D3 domains suitable for insertion or substitution withantigens (e.g., maturational cleavage site, HA, M2e).

FIG. 78 depicts the amino acid sequence of Pseudomonas aeruginosaflagellin (SEQ ID NO: 815). Lysine residues are indicated by theasterisks.

FIG. 79 depicts the amino acid sequence of a S. typhimurium flagellin(SEQ ID NO: 816). Lysine residues are indicated by the asterisks.

FIG. 80 depicts the amino acid sequence (SEQ ID NO: 820) of Listeriamonocytogenes flagellin (GenBank Accession No: Q92DW3). Lysine residuesare indicated by the asterisks.

FIG. 81 depicts a synthetic and manufacturing process for Pam3Cys.M2e.

DETAILED DESCRIPTION OF THE INVENTION

The features and other details of the invention, either as steps of theinvention or as combinations of parts of the invention, will now be moreparticularly described and pointed out in the claims. It will beunderstood that the particular embodiments of the invention are shown byway of illustration and not as limitations of the invention. Theprinciple features of this invention can be employed in variousembodiments without departing from the scope of the invention.

The invention generally is directed to methods of making compositions,and to compositions, that stimulate an immune response in a subject,such as a protective immune response, and methods of treatment, such asby administering the composition to a subject.

“Stimulating an immune response,” as used herein, refers to thegeneration of antibodies and/or T-cells to at least a portion of anantigen, such as the protein portions of hemagglutinin (HA) (e.g., HA1-1, HA 1-2 proteins) described herein. The antibodies and/or T-cellscan be generated to at least a portion of an influenza viral protein(e.g., HA and M2 proteins of influenza A, B and/or C).

Stimulating an immune response in a subject can include the productionof humoral and/or cellular immune responses that are reactive againstthe antigen, such as a viral protein, in particular, an influenza viralprotein.

The compositions of the invention for use in methods to stimulate immuneresponses in subjects, can be evaluated for the ability to stimulate animmune response in a subject using well-established methods. Exemplarymethods to determine whether the compositions of the invention stimulatean immune response in a subject, include measuring the production ofantibodies specific to the antigen (e.g., IgG antibodies) by a suitabletechnique such as, ELISA assays; the potential to induceantibody-dependent enhancement (ADE) of a secondary infection;macrophage-like assays; neutralization assessed by using the PlaqueReduction Neutralization Test (PRNT₈₀); and the ability to generateserum antibodies in non-human models (e.g., mice, rabbits, monkeys)(Putnak, et al., Vaccine 23:4442-4452 (2005)).

“Stimulates a protective immune response,” as used herein, meansadministration of the compositions of the invention, such ashemagglutinin (HA) proteins (e.g., HA1-1, HA1-2 proteins describedherein), results in production of antibodies to the protein to therebycause a subject to survive challenge by an otherwise lethal dose of aviral protein, such as viral HA. Techniques to determine a lethal doseof a virus (e.g., an influenza virus) are known to one of skill in theart (see, for example, WHO/CDS/CSR/NCS2002.5 “WHO Manual on AnimalInfluenza Diagnosis and Surveillance” World Health Organization, Dept ofCommunicable Disease Surveillance and Response, WHO Global InfluenzaProgramme; Harmon, M. W., et al., J. Clin. Microbiol. 26:333-337 (1988);Reed, L. J., et al., Am. J. Hyg. 27:493-497 (1938); Rose, T., et al., J.Clin. Microbiol. 37:937-943 (1999); Walls, H. H. et al., J. Clin.Microbiol. 23:240-245 (1986); Current Protocols in Immunology,19.11.1-19.11.32, Cottey, R., et al., John Wiley & Sons, Inc (2001)).Exemplary techniques for determining a lethal dose can includeadministration of varying doses of virus and a determination of thepercent of subjects that survive following administration of the dose ofvirus (e.g., LD₁₀, LD₂₀, LD₄₀, LD₅₀, LD₆₀, LD₇₀, LD₈₀, LD₉₀). Forexample, a lethal dose of a virus that results in the death of 50% of apopulation of subjects is referred to as an “LD₅₀”; a lethal dose of avirus that results in the death of 80% of a population of subjects isreferred to herein as “LD₈₀”; a lethal dose of a virus that results indeath of 90% of a population of subjects is referred to herein as“LD₉₀.”

For example, determination of the LD₉₀ can be conducted in subjects(e.g., mice) by administering intranasally varying doses (e.g.,dilutions, such as log and half-log dilutions of 8×10³ egg-infectiousdoses (EID)) followed by an assessment of the survival of the subjectsabout 14 days to about 21 days after infection with the virus.Protective immunity can be assessed by physical appearance of thesubject, general demeanor (active), weight (initial loss of weightfollowed by return to a weight about the weight of the subject prior toinfection with the virus) and survival after about 14 to about 21 daysfollowing infection with the virus.

Assessment of stimulation of protective immunity can also be made byemploying assays that assess the ability of the antibodies produced inresponse to the compositions of the invention (e.g., a portion of theprotein of the naturally occurring virus, such as a protein portion ofhemagglutinin) to neutralize binding of the viral protein (e.g.,hemagglutinin protein) to a host cell (see, for example, CurrentProtocols in Immunonology, 19.11.1-19.11.32, Cottey, R., et al., JohnWiley & Sons, Inc (2001)). Assessment of stimulation of protectiveimmunity can also be made by employing assays that measure the abilityof antibodies to inhibit hemagglutinin binding (see, for example,Burnett, F. M., et al., J. exp. Biol. Med. Sci. 25:227-233 (1947); Salk,J. E. J. Immunol. 49:87-98 (1944); Current Protocols in Immunology,19.11.1-19.11.32, Cottey, R., et al., John Wiley & Sons, Inc (2001)).

It is believed that inhibition of hemagglutinin binding is indicative ofthe ability of antibodies, formed from the compositions and by themethods of the invention, to neutralize the sialic acid binding sites ofthe naturally occurring viral hemagglutinin (“neutralization of HAbinding”) and, thereby, prevent infection of the host cell as aconsequence of stimulating a protective immune response Inhibition orneutralization of hemagglutinin binding is believed to correlate with anability of an immune response to protect against a lethal dose of virus.

Neutralization of HA binding can be assessed by in vitro assays (See,for example, Current Protocols in Immunology 19.11.1-19.11.32, Cottey,R., et al., Suppl. 42, John Wiley & Sons, Inc. (2001) and WHO Manual onAnimal Influenza Diagnosis and Surveillance, Webster, R., et al., pages28-36, 48-54, 82-92 (2002)). Exemplary viral neutralization assays relyon the ability of serum to specifically bind and prevent replication ofinfluenza virus in culture, such as in the Madin-Darby Canine Kidney(MDCK) cell line. Briefly, cells are cultured in 96 well plates in thepresence of a previously titered virus and the cytopathic effect of thereplicating virus is observed under a microscope. To test serum, serialdilutions of the serum are prepared and preincubated with the viralstock for 2 hours at 37° C. prior to infecting the MDCK cells. Themixture is incubated for an additional 2 hours after which thevirus/serum mixture is removed and replaced with fresh media. The cellsare grown for 4 days. Wells are scored as positive for viral growth ifat least about 50% of the cells are dead in at least about half of thewells for a given serum dilution. The reciprocal of the highest dilutionof serum which protects at least about half of the cells from death, inat least about half of the wells, is considered the neutralizationtiter.

Alternatively, a micro-neutralization in vitro assay can be performed toassess neutralization of HA binding. For example, serum is diluted andpreincubated with a known titer of virus and mixed with MDCK cells, asdescribed above. After 2 days of incubation, cells are washed and fixedwith acetone. The plates are developed as an ELISA using a monoclonalantibody to the influenza nuclear antigen NP. A microneutralizationtiter is determined as the reciprocal of the highest dilution whichyields less than about 50% of the anti-NP reading of the virus-onlycontrol wells.

The Hemagglutination Inhibition (HAI) assay is based on the HA antigenon the surface of the influenza virus agglutinating red blood cells(RBC) and preventing red blood cells from precipitating. Antibodies thatspecifically bind the sialic acid-binding regions of HA preventagglutination allowing precipitation. The assay is performed in 96 wellV bottom plates with fresh chicken RBC. A stock of viral antigen istitered so that about a 4-fold excess of antigen is present relative tothe minimum amount needed to prevent precipitation. The test serum,which can be from several species including mouse, ferret, poultry orhuman, is heated to about 56° C. to inactivate complement. Serial 2-folddilutions of the inactivated serum are performed and mixed with thestock HA. After about 30 minutes at room temperature, the RBCs are addedand the plate is incubated for about 30 to about 45 minutes. Results arescored by observations: agglutination results in cloudy wells whileinhibition results in a “button” of red cells precipitated at the bottomof the well. Controls include RBC with no HA, which forms a button, andHA and RBC with no serum, which remains cloudy. The HAI titer of aparticular serum sample is the reciprocal of the last dilution whichprevents agglutination (i.e., forms a button). For example, if about a1:128 dilution reads as a button but the 1:256 dilution does not, theHAI titer is about 128.

In one embodiment, the invention is a method of making a protein thatstimulates a protective immune response in a subject, comprising thesteps of separating a portion of a protein from a naturally occurringviral hemagglutinin to thereby make a protein portion, wherein theprotein portion includes at least a portion of a globular head and atleast a portion of at least one secondary structure that causes theglobular head to essentially retain its tertiary structure, and whereinthe protein portion lacks a membrane fusion domain, a transmembranedomain and a cytoplasmic domain. The nucleic acid sequence encoding theprotein portion is transformed into a prokaryotic cell and theprokaryotic host cell is cultured to thereby make the protein thatstimulates a protective immune response in a subject.

“A portion of a protein,” or “protein portion,” as used herein inreference to a naturally occurring viral hemagglutinin, refers to anypart of the naturally occurring viral hemagglutinin that is less thanthe entire naturally occurring viral hemagglutinin. “Naturally occurringviral hemagglutinin,” as used herein, refers to the entire viralhemagglutinin, as it occurs in nature.

The protein portion can further lack a signal sequence. The proteinportion can further include a sialic acid binding site.

Portions of a protein of a naturally occurring viral hemagglutinin foruse in the compositions and methods of the invention can be a portion ofOrthomyxoviridae (influenza A, B, C), Paramyxovirus (parainfluenza,respiratory syncytial virus, Newcastle disease virus, Nipah, Measles,canine distemper, Sendai virus), Reoviridae (rotavirus), Parvoviridae(human parvovirus, porcine parvovirus), Orthopoxvirus (Monkeypox virus,Ectromelia virus), Flaviviridae (West Nile, Japanese Encephalitis, St.Louis, Murray Valley, Kunjin), Avipoxvirus (Chicken fowlpox), Nipahvirus (Guillaume V., et al., J. Virol., 80:7546-54 (2006)); Caninedistemper virus (Singethan K., et al., J Gen Virol., 87:1635-42 (2006));Newcastle disease virus, (de Leeuw O. S., et al., J Gen Virol.,86:1759-69 (2005); and Melanson V. R., et al., J Virol., 78:13053-61(2004); Deng R., et al., Virology, 204:17-26; (1994)), Measles (MasseN., et al., J Virol., 78:9051-63 (2004)), Sendai virus (Tomasi M., etal., FEBS Lett., 11:56-60 (2003)), Human parainfluenza (Porotto M., etal., J Virol., 79:2383-92 (2005); Tsurudome M., et al., Virology,213:190-203 (1995); Bousse T., et al., Virology, 209:654-7 (1995); andTakimoto T., et al., J Virol., 66:7597-600 (1992)).

Portions of a viral hemagglutinin (“protein portions”) (e.g., aninfluenza A, an influenza B and an influenza C viral hemagglutinin) caninclude at least one member selected from the group consisting ofprotein portions referred to herein as “HA1-1,” “HA1-2” and “HA1-3.”

“HA1-1,” as used herein, refers to a protein portion of a viralhemagglutinin that includes at least about one β-sandwich that includesthe substrate binding site, which includes at least about two β-sheets,at least about two to about three short α-helixes, at least one smallβ-sheet and at least one additional small β-sandwich at the bottom ofthe molecule and at least about four disulfide bonds. The β-sandwichthat includes the substrate binding site of the HA1-1 includes at leastabout four β-strands as the top sheet and at least about three to aboutfour β-strands as the bottom sheet. At least about one α-helix of theHA1-1 portion is located by the side of β-sandwich that includes thesubstrate binding site and at least about one to about two are locatedat the bottom of the β-sandwich that includes the substrate bindingsite. The small β-sandwich of the HA1-1 can include at least about twoto about three β-strands in each β-sheet; or about three to about fourβ-strands. Exemplary HA1-1 protein portions include SEQ ID NOS: 8, 11,14, 17, 20, 38, 40, 45, 47, 49, 179, 180, 181 and 182.

“HA1-2,” as used herein, refers to a protein portion of a viralhemagglutinin that includes at least about one β-sandwich that includesthe substrate binding site, at least about two to about three shortα-helixes, at least about one small β-sheet at the bottom of themolecule and at least about two disulfide bonds. A β-strand in a viralhemagglutinin can include between about two to about 15 amino acids. Asmall β-strand can include about two amino acids; or between about twoto about three amino acids; or between about two to four amino acids orbetween about two to about five amino acids. A small β-sheet can includebetween about two to about three β-strands; or between about three toabout four β-strands. The β-sandwich that includes the substrate bindingsite of HA1-2 can further include at least about four β-strands as thetop sheet and at least about three to about four β-strands as the bottomsheet. At least about one α-helix of the HA1-2 portion is located by theside of the β-sandwich that includes the substrate binding site and atleast about one to about two are located at the bottom of the β-sandwichthat includes the substrate binding site. Exemplary HA1-2 proteinportions include SEQ ID NOS: 9, 12, 15, 18, 21, 24, 26, 28, 30, 32, 39,41, 46, 48 and 50.

“HA1-3,” as used herein, refers to a protein portion of a viralhemagglutinin that includes at least one β-sandwich that includes thesubstrate binding site, at least about two short α-helixes and at leastone disulfide bond. “β-sandwich,” as used herein, refers to at leastabout two sets of beta-sheets that form at least about one interactivelayer. “Substrate binding site,” as used herein in reference to theβ-sandwich, means any part of the portion of the naturally occurringviral hemagglutinin that has the capacity to interact or bind to amolecule. For example, the β-sandwich that includes the substratebinding site of the portion can include a portion that binds sialicacid. The β-sandwich that includes the substrate binding site of HA1-3can further include at least about four β-strands as the top sheet andat least about three β strands as the bottom sheet. At least about oneα-helix of the HA1-1 portion is located by the side of the β-sandwichthat includes the substrate binding site and at least one other α-helixis located at the bottom of the β-sandwich that includes the substratebinding site. A short α-helix can include less than about 5 turns (2, 3,4, 5 turns) in an α-helix. An α-helix in a viral hemagglutinin can bebetween one to about 15 turns; or between about two to 15 turns.Exemplary HA1-3 protein portions include SEQ ID NOS: 10, 13, 16, 19, 22,25, 27, 29, 31 and 33.

“A sialic acid binding site,” as that phrase is used herein in referenceto the portion of the protein from the naturally occurring viralhemagglutinin, means a part of the protein portion that has the capacityto interact with sialic acid residues. “A sialic acid binding site” isalso referred to herein as “a sialic acid binding domain.”

“At least a portion,” as used herein, refers to any part of a component(e.g., a globular head, a secondary structure) or molecule (e.g., aprotein, antigen, Toll-like Receptor, a peptide, flagellin, HA, matrix 2protein (M2), matrix 2 ectodomain (M2e)); or the entirety of thecomponent or the molecule. “At least a portion,” is also referred toherein as a “fragment.”

“At least a portion,” as used herein in reference to a flagellin (e.g.,fljB/STF2, E. coli fliC, S. muenchen fliC), refers to any part of theflagellin (e.g., motif C; motif N; domain 1, 2, 3) or the entirety ofthe flagellin that can initiate an intracellular signal transductionpathway for a Toll-like Receptor.

A single polypeptide can exhibit several types of secondary structure.Without any stabilizing interactions, a polypeptide can assume arandom-coil conformation. However, secondary structures, such asalpha(α)-helices and beta(β)-strands, can stabilize a protein or aportion of a protein. Lateral association of β-strands form β-sheets(also referred to herein as “β-pleated sheets”). Secondary structurescan be located at the surfaces of the portion, the protein, or thenaturally occurring protein (e.g., viral hemagglutinin, flagellin, M2e).A tertiary structure of a protein is the three-dimensional arrangementof amino acid residues. In contrast to secondary structure, which isstabilized by, for example, hydrogen bonds, α-helices, β-strands,tertiary structure results from hydrophobic interactions betweennon-polar side chains of the portion, protein or naturally occurringviral hemagglutinin. The hydrophobic interactions hold the helicesstrands in random coils in a compact internal scaffold. The size andshape of a protein can depend on its primary amino acid sequence, aswell as the number, size and arrangement of secondary structures.

“A globular head,” as that phrase is used herein, refers to a portion ofa protein of a naturally occurring viral hemagglutinin that includes thereceptor or sialic acid binding regions. “Globular head,” is alsoreferred to herein as a “globular domain.” The globular head of viralhemagglutinin proteins has been determined based on x-raycrystallography as described, for example, by Wilson I. A., et al.Nature 289:366-373 (1981); Chen, J., et al., Cell 95:409-417 (1998); HaY., et al., The EMBO Journal 21:865-875 (2002); Russell, R. J., et al.,Virology 325:287-296 (2004); and Cox, N. J., et al., In: Toply andWilson's Microbiology and Microbial Infections, eds. B W J Mathy, etal., Vol. 1 (9^(th) ed.) New York, N.Y., Oxford Univ. Press, Ch. 32, p.634 (1998). The globular head of a naturally occurring viralhemagglutinin is a component of the HA1 subunit of, for example,influenza viral hemagglutinin. In addition to the receptor bindingdomain, the globular head can include the E⁻ subdomain and F⁻ subdomainas described, for example, by Ha, Y., et al. The EMBO Journal 21:865-875(2002).

The phrase, “causes the globular head to essentially retain its tertiarystructure,” as used herein, refers to maintenance of the tertiarystructure of the globular head of the naturally occurring viralhemagglutinin sufficient to stimulate a protective immune response in asubject.

The membrane fusion domain of a viral hemagglutinin is that region ofthe viral hemagglutinin (involved in binding of the viral hemagglutinin)that binds a host cell. A transmembrane domain of the viralhemagglutinin is that portion of the viral hemagglutinin that spans themembrane of the virus. A cytoplasmic domain of a viral hemagglutinin isthat portion of the viral hemagglutinin located on the cytoplasmicsurface of the virus.

The portion of the protein of the naturally occurring viralhemagglutinin (also referred to herein as “protein portion”) can furtherlack a signal sequence. The portion of a globular head employed in themethods described herein can include at least a portion of at least onesecondary structure that includes at least a portion of at least oneβ-pleated sheet; at least one alpha helix and/or at least one memberselected from the group consisting of a salt bridge, a leucine zipperand a zinc finger. The portion of a globular head can further include atleast about one disulfide bond, at least about two disulfide bonds, atleast about three disulfide bonds, at least about four disulfide bonds,at least about five disulfide bonds and at least about six disulfidebonds.

The method of making a protein that stimulates a protective immuneresponse in a subject can further include the step of substituting anucleic acid sequence encoding at least one amino acid residue selectedfrom the group consisting of a hydrophilic amino acid residue, a polaramino acid residue and a neutral amino acid residue for a nucleic acidsequence that encodes at least one hydrophobic amino acid residue in theprotein portion. The hydrophobic amino acid residue substituted caninclude at least one member selected from the group consisting of aphenylalanine residue, a tryptophan residue and tyrosine residue. Thepolar amino acid residue substituted for the hydrophobic amino acid caninclude at least one member selected from the group consisting of anaspartic acid residue and a glutamic acid residue.

The portion of a protein of a naturally occurring viral hemagglutinincan be a portion of a naturally occurring influenza viral hemagglutininprotein (e.g., influenza A, B and C). The influenza A viralhemagglutinin protein can be at least one member selected from the groupconsisting of H1, H2, H3, H5, H7 and H9.

The host cell employed in the methods described herein can be aprokaryotic host cell. The prokaryotic host cell can be at least onemember selected from the group consisting of an E. coli prokaryotic hostcell, a Pseudomonas prokaryotic host cell, a Bacillus prokaryotic hostcell, a Salmonella prokaryotic host cell and a P. fluorescensprokaryotic host cell.

The method of making a protein that stimulates a protective immuneresponse in a subject can further include the step of transforming theprokaryotic host cell with a chaperone nucleic acid sequence. Thechaperone nucleic acid sequence can be at least one member selected fromthe group consisting of a groES-groEL chaperone, a dnaK-dnaJ-grpEchaperone, a groES-groEL-tig chaperone and a tig chaperone.

The method of making a protein that stimulates a protective immuneresponse in a subject can further include the step of fusing at least aportion of a Toll-like Receptor (TLR) agonist to the protein. Thenucleic acid sequence encoding at least a portion of a Toll-likeReceptor agonist can be operably linked to the nucleic acid sequenceencoding the protein portion of the viral hemagglutinin. A linker (e.g.,peptide linker) can be between the Toll-like Receptor agonist and theportion of the viral hemagglutinin.

Toll-like Receptors refer to a family of receptor proteins that arehomologous to the Drosophila melangogaster Toll protein. Toll-likeReceptors are type I transmembrane signaling receptor proteinscharacterized by an extracellular leucine-rich repeat domain and anintracellular domain homologous to an interleukin 1 receptor. Toll-likeReceptors include TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR 8, TLR9,TLR10, TLR11 and TLR12.

“Agonist,” as used herein in referring to a TLR, means a molecule thatactivates a TLR signaling pathway. A TLR signaling pathway is anintracellular signal transduction pathway employed by a particular TLRthat can be activated by a TLR ligand or a TLR agonist. Commonintracellular pathways are employed by TLRs and include, for example,NF-κB, Jun N-terminal kinase and mitogen-activated protein kinase. TheToll-like Receptor agonist can include at least one member selected fromthe group consisting of a TLR1 agonist, a TLR2 agonist (e.g., Pam3Cys,Pam2Cys, bacterial lipoprotein), a TLR3 agonist (e.g., dsRNA), a TLR4agonist (e.g., bacterial lipopolysaccharide), a TLR5 agonist (e.g., aflagellin), a TLR6 agonist, a TLR7 agonist, a TLR8 agonist, a TLR9agonist (e.g., unmethylated DNA motifs), TLR10 agonist, a TLR11 agonistand a TLR12 agonist.

The Toll-like Receptor agonists for use in the methods and compositionsof the invention can be a Toll-like Receptor agonist component that isat least a portion of a Toll-like Receptor agonist, wherein theToll-like Receptor agonist component includes at least one cysteineresidue in a position where a cysteine does not occur in the nativeToll-like Receptor agonist, whereby the Toll-like Receptor agonistcomponent activates a Toll-like Receptor.

TLR4 ligands (e.g., TLR4 agonists) for use in the compositions andmethods of the invention can include at least one member selected fromthe group consisting of SEQ ID NOS: 359-406 (see, PCT/US 2006/002906/WO2006/083706; PCT/US 2006/003285/WO 2006/083792; PCT/US 2006/041865;PCT/US 2006/042051).

(SEQ ID NO: 359) GGKSGRTG (SEQ ID NO: 360) KGYDWLVVG (SEQ ID NO: 361)EDMVYRIGVP (SEQ ID NO: 362) VKLSGS (SEQ ID NO: 363) GMLSLALF(SEQ ID NO: 364) CVVGSVR (SEQ ID NO: 365) IVRGCLGW (SEQ ID NO: 366)AAEERTLG (SEQ ID NO: 367) WARVVGWLR (SEQ ID NO: 368) SEGYRLFGG(SEQ ID NO: 369) LVGGVVRRGS (SEQ ID NO: 370) GRVNDLWLAA (SEQ ID NO: 371)SGWMLWREGS (SEQ ID NO: 372) ERMEDRGGDL (SEQ ID NO: 373) KLCCFTECM(SEQ ID NO: 374) AVGSMERGRG (SEQ ID NO: 375) RDWVGGDLV (SEQ ID NO: 376)FFEVAKISQQ (SEQ ID NO: 377) WWYWC (SEQ ID NO: 378) MHLCSHA(SEQ ID NO: 379) WLFRRIG (SEQ ID NO: 380) YWFWRIG (SEQ ID NO: 381)MHLYCIA (SEQ ID NO: 382) WPLFPWIV (SEQ ID NO: 383) DMRSHAR(SEQ ID NO: 384) MHLCTHA (SEQ ID NO: 385) NLFPFY (SEQ ID NO: 386)MHLCTRA (SEQ ID NO: 387) RHLWYHA (SEQ ID NO: 388) WPFSAYW(SEQ ID NO: 389) WYLRGS (SEQ ID NO: 390) GKGTDLG (SEQ ID NO: 391) IFVRMR(SEQ ID NO: 392) WLFRPVF (SEQ ID NO: 393) FLGWLMG (SEQ ID NO: 394)MHLWHHA (SEQ ID NO: 395) WWFPWKA (SEQ ID NO: 396) WYLPWLG(SEQ ID NO: 397) WPFPRTF (SEQ ID NO: 398) WPFPAYW (SEQ ID NO: 399)FLGLRWL (SEQ ID NO: 400) SRTDVGVLEV (SEQ ID NO: 401) REKVSRGDKG(SEQ ID NO: 402) DWDAVESEYM (SEQ ID NO: 403) VSSAQEVRVP (SEQ ID NO: 404)LTYGGLEALG (SEQ ID NO: 405) VEEYSSSGVS (SEQ ID NO: 406) VCEVSDSVMA

TLR2 ligands (e.g., TLR2 agonists) for use in the compositions andmethods of the invention can also include at least one member selectedfrom the group consisting of SEQ ID NOS: 455-494 (see, PCT/US2006/002906/WO 2006/083706; PCT/US 2006/003285/WO 2006/083792; PCT/US2006/041865; PCT/US 2006/042051).

(SEQ ID NO: 455) NPPTT (SEQ ID NO: 456) MRRIL (SEQ ID NO: 457) MISS(SEQ ID NO: 458) RGGSK (SEQ ID NO: 459) RGGF (SEQ ID NO: 460) NRTVF(SEQ ID NO: 461) NRFGL (SEQ ID NO: 462) SRHGR (SEQ ID NO: 463) IMRHP(SEQ ID NO: 464) EVCAP (SEQ ID NO: 465) ACGVY (SEQ ID NO: 466) CGPKL(SEQ ID NO: 467) AGCFS (SEQ ID NO: 468) SGGLF (SEQ ID NO: 469) AVRLS(SEQ ID NO: 470) GGKLS (SEQ ID NO: 471) VSEGV (SEQ ID NO: 472) KCQSF(SEQ ID NO: 473) FCGLG (SEQ ID NO: 474) PESGV (SEQ ID NO: 475) DPDSG(SEQ ID NO: 476) IGRFR (SEQ ID NO: 477) MGTLP (SEQ ID NO: 478) ADTHQ(SEQ ID NO: 479) HLLPG (SEQ ID NO: 480) GPLLH (SEQ ID NO: 481) NYRRW(SEQ ID NO: 482) LRQGR (SEQ ID NO: 483) IMWFP (SEQ ID NO: 484) RVVAP(SEQ ID NO: 485) IHVVP (SEQ ID NO: 486) MFGVP (SEQ ID NO: 487) CVWLQ(SEQ ID NO: 488) IYKLA (SEQ ID NO: 489) KGWF (SEQ ID NO: 490) KYMPH(SEQ ID NO: 491) VGKND (SEQ ID NO: 492) THKPK (SEQ ID NO: 493) SHIAL(SEQ ID NO: 494) AWAGT

The TLR2 ligand (e.g., TLR2 agonist) can also include at least a portionof at least one member selected from the group consisting of flagellinmodification protein FlmB of Caulobacter crescentus; Bacterial Type IIIsecretion system protein; invasin protein of Salmonella; Type 4 fimbrialbiogenesis protein (PilX) of Pseudomonas; Salmonella SciJ protein;putative integral membrane protein of Streptomyces; membrane protein ofPseudomonas; adhesin of Bordetella pertusis; peptidase B of Vibriocholerae; virulence sensor protein of Bordetella; putative integralmembrane protein of Neisseria meningitidis; fusion of flagellarbiosynthesis proteins FliR and FlhB of Clostridium; outer membraneprotein (porin) of Acinetobacter; flagellar biosynthesis protein FlhF ofHelicobacter; ompA related protein of Xanthomonas; omp2a porin ofBrucella; putative porin/fimbrial assembly protein (LHrE) of Salmonella;wbdk of Salmonella; Glycosyltransferase involved in LPS biosynthesis;Salmonella putative permease.

The TLR2 ligand (e.g., TLR agonist) can include at least a portion of atleast one member selected from the group consisting oflipoprotein/lipopeptides (a variety of pathogens); peptidoglycan(Gram-positive bacteria); lipoteichoic acid (Gram-positive bacteria);lipoarabinomannan (mycobacteria); a phenol-soluble modulin(Staphylococcus epidermidis); glycoinositolphospholipids (TrypanosomaCruzi); glycolipids (Treponema maltophilum); porins (Neisseria); zymosan(fungi) and atypical LPS (Leptospira interrogans and Porphyromonasgingivalis).

The TLR2 ligand (e.g., TLR2 agonist) can also include at least onemember selected from the group consisting of SEQ ID NOS: 495-497 (see,PCT/US 2006/002906/WO 2006/083706; PCT/US 2006/003285/WO 2006/083792;PCT/US 2006/041865; PCT/US 2006/042051).

(SEQ ID NO: 495) KGGVGPVRRSSRLRRTTQPG (SEQ ID NO: 496)GRRGLCRGCRTRGRIKQLQSAHK (SEQ ID NO: 497) RWGYHLRDRKYKGVRSHKGVPR

The TLR2 agonist can include at least a portion of a bacteriallipoprotein (BLP).

The TLR2 agonist can be a bacterial lipoprotein, such as Pam2Cys(S—[2,3-bis(palmitoyloxy) propyl]cysteine), Pam3Cys([Palmitoyl]-Cys((RS)-2,3-di(palmitoyloxy)-propyl cysteine) orPseudomonas aeruginosa OprI lipoprotein (OprI). Exemplary OprIlipoproteins include SEQ ID NO: 782, encoded by SEQ ID NO: 783. Anexemplary E. coli bacterial lipoprotein for use in the inventiondescribed herein is SEQ ID NO: 784 encoded by SEQ ID NO: 785. Abacterial lipoprotein that activates a TLR2 signaling pathway (a TLR2agonist) is a bacterial protein that includes a palmitoleic acid(Omueti, K. O., et al., J. Biol. Chem. 280: 36616-36625 (2005)). Forexample, expression of SEQ ID NOS: 783 and 785 in bacterial expressionsystems (e.g., E. coli) results in the addition of a palmitoleic acidmoiety to a cysteine residue of the resulting protein (e.g., SEQ ID NOS:782 and 784) thereby generating a TLR2 agonist for use in thecompositions, fusion proteins and polypeptides of the invention.Production of tripalmitoylated-lipoproteins (also referred to astriacyl-lipoproteins) in bacteria occurs through the addition of adiacylglycerol group to the sulfhydryl group of a cysteine (e.g.,cysteine 21 of SEQ ID NO: 784) followed by cleavage of the signalsequence and addition of a third acyl chain to the free N-terminal groupof the same cysteine (e.g., cysteine 21 of SEQ ID NO: 784) (Sankaran,K., et al., J. Biol. Chem. 269:19706 (1994)), to generate atripalmitylated peptide (a TLR2 agonist) as shown, for example, in FIG.74.

The Toll-like Receptor agonist in the compositions of the invention canfurther include at least one cysteine residue at the terminal amino acidof the amino-terminus and/or the terminal amino acid of thecarboxy-terminus of the Toll-like Receptor agonist. For example, SEQ IDNO: 359 can further include at least one cysteine residue in a peptidebond to the amino-terminal glycine residue and/or at least one cysteineresidue in a peptide bond to the carboxy-terminal glycine residue; SEQID NO: 360 can further include at least one cysteine residue in apeptide bond to the amino-terminal lysine residue and/or at least onecysteine residue in a peptide bond to the carboxy-terminal glycineresidue; SEQ ID NO: 361 can further include at least one cysteineresidue in a peptide bond to the amino-terminal glutamic acid residueand/or at least one cysteine residue in a peptide bond to thecarboxy-terminal proline residue.

TLR5 agonists for use in the methods of the invention can include atleast a portion of a flagellin. Flagellin are pathogen-associatedmolecular patterns (PAMPs) that activate TLR5.

The flagellin in the compositions and methods described herein can be atleast a portion of a S. typhimurium flagellin (Genbank Accession NumberAF045151); at least a portion of the S. typhimurium flagellin selectedfrom the group consisting of SEQ ID NO: 498, SEQ ID NO: 812, SEQ ID NO:816 and SEQ ID NO: 500; at least a portion of an S. muenchen flagellin(Genbank Accession Number AB028476) that includes at least a portion ofSEQ ID NO: 504 and SEQ ID NO: 813; at least a portion of P. aeruginosaflagellin that includes at least a portion of SEQ ID NO: 815; at least aportion of a Listeria monocytogenes flagellin that includes at least aportion of SEQ ID NO: 820; at least a portion of an E. coli flagellinthat includes at least a portion of SEQ ID NO: 502 and SEQ ID NO: 814;at least a portion of a Yersinia flagellin; and at least a portion of aCampylobacter flagellin.

The flagellin employed in the compositions of the invention can alsoinclude the polypeptides of SEQ ID NO: 498, SEQ ID NO: 500, SEQ ID NO:504 and SEQ ID NO: 502; at least a portion of SEQ ID NO: 498, at least aportion of SEQ ID NO: 500, at least a portion of SEQ ID NO: 504 and atleast a portion of SEQ ID NO: 502; and a polypeptide encoded by SEQ IDNO: 499, SEQ ID NO: 501, SEQ ID NO: 505 and SEQ ID NO: 503; or at leasta portion of a polypeptide encoded by SEQ ID NO: 499, SEQ ID NO: 501,SEQ ID NO: 505 and SEQ ID NO: 503.

The flagellin employed in the compositions and method of the inventioncan lack at least a portion of a hinge region. Hinge regions are thehypervariable regions of a flagellin. Hinge regions of a flagellin arealso referred to herein as “D3 domain or region,” “propellor domain orregion,” “hypervariable domain or region” and “variable domain orregion.” “Lack” of a hinge region of a flagellin, means that at leastone amino acid or at least one nucleic acid codon encoding at least oneamino acid that comprises the hinge region of a flagellin is absent inthe flagellin. Examples of hinge regions include amino acids 176-415 ofSEQ ID NO: 498, which are encoded by nucleic acids 528-1245 of SEQ IDNO: 499; amino acids 174-422 of SEQ ID NO: 502, which are encoded bynucleic acids 522-1266 of SEQ ID NO: 503; or amino acids 173-464 of SEQID NO: 504, which are encoded by nucleic acids 519-1392 of SEQ ID NO:505. Thus, if amino acids 176-415 were absent from the flagellin of SEQID NO: 498, the flagellin would lack a hinge region. A flagellin lackingat least a portion of a hinge region is also referred to herein as a“truncated version” of a flagellin.

“At least a portion of a hinge region,” as used herein, refers to anypart of the hinge region of the flagellin, or the entirety of the hingeregion. “At least a portion of a hinge region” is also referred toherein as a “fragment of a hinge region.” At least a portion of thehinge region of fljB/STF2 can be, for example, amino acids 200-300 ofSEQ ID NO: 498. Thus, if amino acids 200-300 were absent from SEQ ID NO:498, the resulting amino acid sequence of STF2 would lack at least aportion of a hinge region.

Alternatively, at least a portion of a naturally occurring flagellin canbe replaced with at least a portion of an artificial hinge region.“Naturally occurring,” in reference to a flagellin amino acid sequence,means the amino acid sequence present in the native flagellin (e.g., S.typhimurium flagellin, S. muenchin flagellin, E. coli flagellin). Thenaturally occurring hinge region is the hinge region that is present inthe native flagellin. For example, amino acids 176-415 of SEQ ID NO:498, amino acids 174-422 of SEQ ID NO: 502 and amino acids 173-464 ofSEQ ID NO: 504, are the amino acids corresponding to the natural hingeregion of STF2, E. coli fliC and S. muenchen flagellins, fliC,respectively. “Artificial,” as used herein in reference to a hingeregion of a flagellin, means a hinge region that is inserted in thenative flagellin in any region of the flagellin that contains orcontained the native hinge region.

The hinge region of a flagellin be deleted and replaced with an antigen(e.g., HA1-1, HA1-2, the maturational cleavage site) and the resultingconstruct fused to another antigen (e.g., 4×M2e).

An artificial hinge region may be employed in a flagellin that lacks atleast a portion of a hinge region, which may facilitate interaction ofthe carboxy- and amino-terminus of the flagellin for binding to TLR5and, thus, activation of the TLR5 innate signal transduction pathway. Aflagellin lacking at least a portion of a hinge region is designated bythe name of the flagellin followed by a “Δ.” For example, an STF2 (e.g.,SEQ ID NO: 498) that lacks at least a portion of a hinge region isreferenced to as “STF2Δ” or “fljB/STF2Δ” (e.g., SEQ ID NO: 500).

The flagellin for use in the methods and compositions of the inventioncan be a at least a portion of a flagellin, wherein the flagellincomponent includes at least one cysteine residue and whereby theflagellin component activates a Toll-like Receptor 5; a flagellincomponent that is at least a portion of a flagellin, wherein at leastone lysine of the flagellin component has been substituted with at leastone arginine, whereby the flagellin component activates a Toll-likeReceptor 5; a flagellin component that is at least a portion of aflagellin, wherein at least one lysine of the flagellin component hasbeen substituted with at least one serine residue, whereby the flagellincomponent activates a Toll-like Receptor 5; a flagellin component thatis at least a portion of a flagellin, wherein at least one lysine of theflagellin component has been substituted with at least one histidineresidue, whereby the flagellin component activates a Toll-like Receptor5, as described herein.

A recombinant fusion protein can be generated by operably linking a TLRagonist to the protein portion (e.g., HA1-1, HA1-2).

“Fusion protein,” as used herein, refers to a protein generated from atleast two similar or distinct components (e.g., a protein portion of HAand a TLR agonist). Fusion proteins can be generated recombinantly or bychemical conjugation.

In an embodiment, fusion proteins of protein portions of HA and TLRagonists (e.g., SEQ ID NOS: 89-92, 95, 151-160, 177, 209, 210 and 211)can be admixed or coadministered with fusion proteins of TLR agonistsand M2e proteins (e.g., SEQ ID NOS: 528, 587, 589 and 591) andadministered to a subject to stimulate an immune response, such as aprotective immune response.

Fusion proteins of the invention can be designated by components of thefusion proteins separated by a “.”. For example, “STF2.HA1-2” refers toa fusion protein comprising one STF2 protein and one HA1-2 protein; and“STF2Δ.HA1-2” refers to a fusion protein comprising one STF2 proteinwithout the hinge region and HA1-2. Exemplary fusion proteins of theinvention include SEQ ID NOS: 89-92, 95, 151-160, 177, 209, 210 and 211.

The fusion proteins can include, for example, two, three, four, five,six or more Toll-like Receptor agonists (e.g., flagellin) and two,three, four, five, six or more antigens (e.g., protein portions, such asHA1-1, HA1-2). When two or more TLR agonists and/or two or more proteinportions comprise fusion proteins of the invention, they are alsoreferred to as “multimers.” For example, a multimer of HA1-1 can be fourHA1-1 sequences, which is referred to herein as 4×HA1-1. Likewise,“2×HA1-1” is a multimer of two HA1-1 sequences. (See, for example, SEQID NOS: 342-346 and 348).

The fusion proteins of the invention can further include a linkerbetween at least one component of the fusion protein (e.g., TLR agonist)and at least one other component of the fusion protein (e.g., HA1-1,HA1-2) of the composition, a linker between at least two of similarcomponents of the fusion protein (e.g., HA1-1, HA1-2) or any combinationthereof “Linker,” as used herein in reference to a fusion protein of theinvention, refers to a connector between components of the fusionprotein in a manner that the components of the fusion protein are notdirectly joined. For example, one part of the fusion protein (e.g.,flagellin) can be linked to a distinct part (e.g., protein portion,HA1-1, HA1-2, an antigen) of the fusion protein. Likewise, at least twoor more similar or like components of the fusion protein can be linked(e.g., two flagellin can further include a linker between eachflagellin, or two HA proteins can further include a linker between eachHA protein).

Additionally, or alternatively, the fusion proteins of the invention caninclude a combination of a linker between distinct components of thefusion protein and similar or like components of the fusion protein. Forexample, a fusion protein can comprise at least two TLR agonists thatfurther includes a linker between, for example, two or more flagellin;at least two protein portions of HA that further include a linkerbetween them; a linker between one component of the fusion protein(e.g., flagellin) and another distinct component of the fusion protein(e.g., protein portions of HA), or any combination thereof.

The linker can be an amino acid linker. The amino acid linker caninclude synthetic or naturally occurring amino acid residues. The aminoacid linker employed in the fusion proteins of the invention can includeat least one member selected from the group consisting of a lysineresidue, a glutamic acid residue, a serine residue and an arginineresidue. The amino acid linker can include, for example, SEQ ID NOS:521, 523, 524 and 526, encoded by the nucleic acid sequences of SEQ IDNOS: 520, 522, 525 and 527 respectively.

The Toll-like Receptor agonist of the fusion proteins of the inventioncan be fused to a carboxy-terminus, the amino-terminus or both thecarboxy- and amino-terminus of the protein portion of HA (e.g., HA1-1,HA1-2 or other antigens, such as the ectodomain of the Matrix 2 protein,a maturational cleavage site).

Fusion proteins can be generated by fusing the protein portions of HA(or other antigens, such as the maturational cleavage site of HA) to atleast one of four regions (Regions 1, 2, 3 and 4) of flagellin, whichhave been identified based on the crystal structure of flagellin(PDB:1UCU) (see FIG. 77).

Region 1 is TIAL (SEQ ID NO: 823) . . . - . . . GLG (95-111 of SEQ IDNO: 841). The corresponding residues for Salmonella typhimurium fljBconstruct are TTLD (SEQ ID NO: 824) . . . - . . . GTN (196-216 of SEQ IDNO: 841). This region is an extended peptide sitting in a groove of twobeta strands (GTDQKID (SEQ ID NO: 825) and NGEVTL (SEQ ID NO: 826) of(SEQ ID NO: 841). Substitution of this peptide with an antigen (e.g., HAmaturational cleavage site) may mimic the conformation of wild typematurational cleavage site peptide in HA0. Exemplary amino acids thatmay be substituted include: flagellin residues SGLDDAAIKAAT (SEQ ID NO:827) (201-212 of SEQ ID NO: 841) substituted with maturational cleavagesite residues: RGIFGAIAGFIE (SEQ ID NO: 828), which correspond to theA/H3N2 subtype, or RGLFGAIAGFIE (SEQ ID NO: 803), which correspond tothe maturational cleavage site residues from the A/H2N1, A/H1N1, A/H5N1subtypes or with RGFFGAIAGFLE (SEQ ID NO: 805), which correspond toInfluenza B HA maturational cleavage site residues.

Region 2 of the Salmonella flagellin is a small loop GTG (238-240 of SEQID NO: 841) in 1UCU structure (see FIG. 77). The corresponding loop inSalmonella fljB is GADAA (SEQ ID NO: 829) (244-248 of SEQ ID NO: 841).Insertion of an antigen (e.g., protein portions of HA, a maturationcleavage site peptide) in this loop or replacement of the entire loopwith an antigen (e.g., protein portions of HA, a maturational cleavagesite peptide) should preserve the extended loop structure of thematurational cleavage site peptide that is associated with the native HAmolecule.

Region 3 is a bigger loop that resides on the opposite side of theRegion 1 peptide (see FIG. 77). This loop can be simultaneouslysubstituted together with region 1 to create a double copy of theantigen (e.g., protein portions of HA, a maturational cleavage sitepeptide). The loop starts from AGGA (SEQ ID NO: 830) and ends at PATA(SEQ ID NO: 831) (259-274 of SEQ ID NO: 841). The correspondingSalmonella fljB sequence is AAGA (SEQ ID NO: 832 . . . - . . . ATTK (SEQID NO: 833) (266-281 of SEQ ID NO: 841). The sequence AGATKTTMPAGA (SEQID NO: 834) (267-278 of SEQ ID NO: 841) can be replaced with theantigens (e.g., protein portions of HA, a maturation cleavage sitepeptides).

Region 4 is the loop (VTGTG (SEQ ID NO: 835)) connecting a short α-helix(TEAKAALTAA (SEQ ID NO: 836)) and a β-strand (ASVVKMSYTDN (SEQ ID NO:837) in 1UCU structure (see FIG. 77). The corresponding loop inSalmonella fljB is a longer loop VDATDANGA (SEQ ID NO: 838 (307-315 ofSEQ ID NO: 841). An antigen (e.g., a protein portion of HA, a maturationcleavage site peptide) can be inserted into or replace this region.

One or more copies of antigens (e.g., maturation cleavage site) can beinserted or used to replace the peptides listed in the above fourregions. Preferably, the replacements would be in Region 1 and Region 3.

Exemplary sequences of maturation cleavage site peptides of HA arelisted below:

Sequence Subtype NVPEKQTRGIFGAIAGFIE A/H3N2 (SEQ ID NO: 800)NVPQIESRGLFGAIAGFIE A/H2N1 (SEQ ID NO: 801) NIPSIQSRGLFGAIAGFIEA/H1N1 (SEQ ID NO: 802) RERRRKKRGLFGAIAGFIE A/H5N1 (SEQ ID NO: 839)PAKLLKERGFFGAIAGFLE B/HA (SEQ ID NO: 804)Sequence alignment of X-ray model (1UCU) and SEQ ID NO: 84175.1% identity in 506 residues overlap; Score: 1703.0; Gap frequency: 2.6%1UCU   1 AQVINTNSLSLLTQNNLNKSQSALGTAIERLSSGLRINSAKDDAAGQAIANRFTANIKGLSfla   2 AQVINTNSLSLLTQNNLNKSQSALGTAIERLSSGLRINSAKDDAAGQAIANRFTANIKGL    ************************************************************ 1UCU 61 TQASRNANDGISIAQTTEGALNEINNNLQRVRELAVQSANSTNSQSDLDSIQAEITQRLN Sfla 62 TQASRNANDGISIAQTTEGALNEINNNLQRVRELAVQSANSTNSQSDLDSIQAEITQRLN    ************************************************************ 1UCU121 EIDRVSGQTQFNGVKVLAQDNTLTIQVGANDGETIDIDLKQINSQTLGLDTLNVQQKYKV Sfla122 EIDRVSGQTQFNGVKVLAQDNTLTIQVGANDGETIDIDLKQINSQTLGLDSLNVQKAYDV    ************************************************** ****  * * 1UCU181 SDTAATVTGYAD--TTI---ALDNSTFKASATGLGGTDQKIDGDLKFDDTTGKYYAKVTV Sfla182 KDTAVTTKAYANNGTTLDVSGLDDAAIKAATGGTNGTASVTGGAVKFDADNNKYFVTIGG     *** *   **   **     **    **   *  **     *  ***    ** 1UCU236 TGGT-GKDGYYEVSVDKTNGEVTLAGGATSPLTGGLPATATEDVKNVQVANADLTEAKA Sfla242 FTGADAAKNGDYEVNV-ATDGTVTLAAGATKTTMPAGATTKTEVQELKDTPAVVSADAKN      *    * * *** *  * * **** ***         * **              ** 1UCU294 ALTAAGVTGT----ASVVKMSYTDNNGKTIDGGLAVKVGDDYYSATQNK-DGSISINTTK Sfla301 ALIAGGVDATDANGAELVKMSYTDKNGKTIEGGYALKAGDKYYAADYDEATGAIKAKTTS    ** * **  *    *  ******* ***** ** * * ** ** *      * *   ** 1UCU349 YTADDGTSKTALNKLGGADGKTEVVSIGGKTYAASKAEGHNFKAQPDLAEAAATTTENPL Sfla361 YTAADGTTKTAANQLGGVDGKTEVVTIDGKTYNASKAAGHDFKAQPELAEAAAKTTENPL    *** *** *** * *** ******* * **** **** ** ***** ****** ****** 1UCU409 QKIDAALAQVDTLRSDLGAVQNRFNSAITNLGNTVNNLTSVRSRIEDSDYATEVSNMSRA Sfla421 QKIDAALAQVDALRSDLGAVQNRFNSAITNLGNTVNNLSEARSRIEDSDYATEVSNMSRA    *********** **************************   ******************* 1UCU469 QILQQAGTSVLAQANQVPQNVLSLLR (SEQ ID NO: 840) Sfla481 QILQQAGTSVLAQANQVPQNVLSLLR (SEQ ID NO: 841)    **************************

The methods of making a protein that stimulates a protective immuneresponse in a subject can further include the step of operably linking anucleic acid sequence encoding a carrier protein to the nucleic acidsequence encoding a portion of the viral hemagglutinin.

“Carrier,” as used herein, refers to a molecule (e.g., protein, peptide)that can enhance stimulation of a protective immune response. Carrierscan be physically attached (e.g., linked by recombinant technology,peptide synthesis, chemical conjugation or chemical reaction) to acomposition (e.g., a protein portion of a naturally occurring viralhemagglutinin) or admixed with the composition.

Carriers for use in the methods and compositions described herein caninclude, for example, at least one member selected from the groupconsisting of Tetanus toxoid (TT), Vibrio cholerae toxoid, Diphtheriatoxoid (DT), a cross-reactive mutant (CRM) of diphtheria toxoid, E. colienterotoxin, E. coli B subunit of heat labile enterotoxin (LTB), Tobaccomosaic virus (TMV) coat protein, protein Rabies virus (RV) envelopeprotein (glycoprotein), thyroglobulin (Thy), heat shock protein HSP 60Kda, Keyhole limpet hemocyamin (KLH), an early secreted antigentuberculosis-6 (ESAT-6), exotoxin A, choleragenoid, hepatitis B coreantigen, and the outer membrane protein complex of N. meningiditis(OMPC) (see, for example, Schneerson, R., et al., Prog Clin Biol Res47:77-94 (1980); Schneerson, R., et al., J Exp Med 152:361-76 (1980);Chu, C., et al., Infect Immun 40: 245-56 (1983); Anderson, P., InfectImmun 39:233-238 (1983); Anderson, P., et al., J Clin Invest 76:52-59(1985); Fenwick, B. W., et al., 54:583-586 (1986); Que, J. U., et al.Infect Immun 56:2645-9 (1988); Que, J. U., et al. Infect Immun 56:2645-9(1988); (Que, J. U., et al. Infect Immun 56:2645-9 (1988); Murray, K.,et al., Biol Chem 380:277-283 (1999); Fingerut, E., et al., Vet ImmunolImmunopathol 112:253-263 (2006); and Granoff, D. M., et al., Vaccine11:Suppl 1:S46-51 (1993)).

Exemplary carrier proteins for use in the methods and compositionsdescribed herein can include at least one member selected from the groupconsisting of SEQ ID NOS: 788-795:

Cross-reactive mutant (CRM) of diphtheria toxin including, CRM197(SEQ ID NO: 788) GADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDDWKGFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEYINNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRRSVGSSLSCINLDWDVIRDKTKTKIESLKEHGPIKNKMSESPNKTVSEEKAKQYLEEFHQTALEHPELSELKTVTGTNPVFAGANYAAWAVNVAQVIDSETADNLEKTTAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMVAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSYNRPAYSPGHKTQPFLHDGYAVSWNTVEDSIIRTGFQGESGHDIKITAENTPLPIAGVLLPTIPGKLDVNKSKTHISVNGRKIRMRCRAIDGDVTFCRPKSPVYVGNGVHANLHVAFHRSSSEKIHSNEISSDSIGVLGYQKTVDHTKVNSKLSLFFEIKSCoat protein of Tobacco mosaic virus (TMV) coat protein (SEQ ID NO: 789)MMAYSIPTPSQLVYFTENYADYIPFVNRLINARSNSFQTQSGRDELREILIKSQVSVVSPISRFPAEPAYYIYLRDPSISTVYTALLQSTDTRNRVIEVENSTNVTTAEQLNAVRRTDDASTAIHNNLEQLLSLLTNGTGVFNRTSF ESASGLTWLVTTTPRTACoat protein of alfalfa mosaic virus (AMV) (SEQ ID NO: 790)MSSSQKKAGGKAGKPTKRSQNYAALRKAQLPKPPALKVPVAKPTNTILPQTGCVWQSLGTPLSLSSSNGLGARFLYSFLKDFAAPRILEEDLIFRMVFSITPSHAGSFCLTDDVTTEDGRAVAHGNPMQEFPHGAFHANEKFGFELVFTAPTHAGMQNQNFKHSYAVALCLDFDALPEGSRNPSYRFNEVWVERKAFPRAGPLRSLITVGLFDDADDLDRQ Coat protein of Potato virus X(SEQ ID NO: 791) MTTPANTTQATGSTTSTTTKTAGATPATTSGLFTIPDGEFFSTARAIVASNAVATNEDLSKIEAIWKDMKVPTDTMAQAAWDLVRHCADVGSSAQTEMIDTGPYSNGISRARLAAAIKEVCTLRQFCMKYAPVVWNWMLTNNSPPANWQAQGFKPEHKFAAFDFFNGVTNPAAIMPKEGLIRPPSEAEMNAAQTAAFVKITKARAQSNDFASLDAAVTRGRITGTTTAEAVVTLPPPPorins from Neisseria sp, e.g.,class I outer membrane protein of Neisseria meningitides(SEQ ID NO: 792) MRKKLTALVLSALPLAAVADVSLYGEIKAGVEGRNYQLQLTEAQAANGGASGQVKVTKVTKAKSRIRTKISDFGSFIGFKGSEDLGEGLKAVWQLEQDVSVAGGGATQWGNRESFIGLAGEFGTLRAGRVANQFDDASQAIDPWDSNNDVASQLGIFKRHDDMPVSVRYDSPEFSGFSGSVQFVPAQNSKSAYKPAYWTTVNTGSATTTTFVPAVVGKPGSDVYYAGLNYKNGGFAGNYAFKYARHANVGRDAFELFLLGSGSDQAKGTDPLKNHQVHRLTGGYEEGGLNLALAAQLDLSENGDKTKNSTTEIAATASYRFGNAVPRISYAHGFDFIERGKKGENTSYDQIIAGVDYDFSKRTSAIVSGAWLKRNTGIGNYTQINAASVGLR HKFMajor fimbrial subunit protein type I (Fimbrillin) (SEQ ID NO: 793)MVLKTSNSNRAFGVGDDESKVAKLTVMVYNGEQQEAIKSAENATKVEDIKCSAGQRTLVVMANTGAMELVGKTLAEVKALTTELTAENQEAAGLIMTAEPKTIVLKAGKNYIGYSGTGEGNHIENDPLKIKRVHARMAFTEIKVQMSAAYDNIYTFVPEKIYGLIAKKQSNLFGATLVNADANYLTGSLTTFNGAYTPANYANVPWLSRNYVAPAADAPQGFYVLENDYSANGGTIHPTILCVYGKLQKNGADLAGADLAAAQAANWVDAEGKTYYPVLVNFNSNNYTYDSNYTPKNKIERNHKYDIKLTITGPGTNNPENPITESAHLNVQCTVAEWVLVGQ NATWMycoplasma fermentans macrophage activating lipopeptide (MALP-2)(SEQ ID NO: 794) MKKSKKILLGLSPIAAVLPAVAVSCGNNDESNISFKEKDISKYTTTNANGKQVVKNAELLKLKPVLITDEGKIDDKSFNQSAFEALKAINKQTGIEINSVEPSSNFESAYNSALSAGHKIWVLNGFKHQQSIKQYIDAHREELERNQIKIIGIDFDIETEYKWFYSLQFNIKESAFTTGYAIASWLSEQDESKRVVASFGVGAFPGVTTFNEGFAKGILYYNQKHKSSKIYHTSPVKLDSGFTAGEKMNTVINNVLSSTPADVKYNPHVILSVAGPATFETVRLANKGQYVIGVDSDQGMIQDKDRILTSVLKHIKQAVYETLLDLILEKEEGYKPYVVKDKKADKKWSHFGTQKEKWIGVAENHFSNTEEQAKINNKIKEAIKMFKELPEDFVKYINSDKALKDGNKIDNVSERLEAIISAINKAAKp19 protein of Mycobacterium tuberculosis (SEQ ID NO: 795)ATTLPVQRHPRSLFPEFSELFAAFPSFAGLRPTFDTRLMRLEDEMKEGRYEVRAELPGVDPDKDVDIMVRDGQLTIKAERTEQKDFDGRSEFAYGSFVRTVSLPVGADEDDIKATYDKGILTVSVAVSEGKPTEKHIQIRSTN

The compositions of the invention can further include at least oneadjuvant. Adjuvants contain agents that can enhance the immune responseagainst substances that are poorly immunogenic on their own (see, forexample, Immunology Methods Manual, vol. 2, I. Lefkovits, ed., AcademicPress, San Diego, Calif., 1997, ch. 13). Immunology Methods Manual isavailable as a four volume set, (Product Code Z37, 435-0); on CD-ROM,(Product Code Z37, 436-9); or both, (Product Code Z37, 437-7). Adjuvantscan be, for example, mixtures of natural or synthetic compounds that,when administered with compositions of the invention, such as proteinsthat stimulate a protective immune response made by the methodsdescribed herein, further enhance the immune response to the protein.Compositions that further include adjuvants may further increase theprotective immune response stimulated by compositions of the inventionby, for example, stimulating a cellular and/or a humoral response (i.e.,protection from disease versus antibody production). Adjuvants can actby enhancing protein uptake and localization, extend or prolong proteinrelease, macrophage activation, and T and B cell stimulation. Adjuvantsfor use in the methods and compositions described herein can be mineralsalts, oil emulsions, mycobacterial products, saponins, syntheticproducts and cytokines Adjuvants can be physically attached (e.g.,linked by recombinant technology, by peptide synthesis or chemicalreaction) to a composition described herein or admixed with thecompositions described herein.

In another embodiment, the invention is a method of making a proteinthat stimulates a protective immune response in a subject, comprisingthe steps of separating a portion of a protein from a naturallyoccurring viral hemagglutinin to thereby form a protein portion, whereinthe protein portion includes at least a portion of a globular head andat least a portion of at least one secondary structure that causes theglobular head to essentially retain its tertiary structure, and whereinthe protein portion lacks a membrane fusion domain, a transmembranedomain and a cytoplasmic domain; transfecting a nucleic acid sequenceencoding the protein portion into a eukaryotic host cell, wherein theeukaryotic host cell is not a Pichia pastoris eukaryotic host cell; andculturing the eukaryotic host cell to thereby make the protein. In oneembodiment, the protein portion can further lack a signal sequence. Inanother embodiment, the protein portion can further include a sialicacid binding site.

In a further embodiment, the invention is a method of making a proteinthat stimulates a protective immune response in a subject, comprisingthe steps of separating a portion of a protein from a naturallyoccurring viral hemagglutinin to thereby form a protein portion, whereinthe protein portion includes at least a portion of a globular head, andat least a portion of at least one secondary structure that causes theglobular head to essentially retain its tertiary structure, and whereinthe protein portion lacks a membrane fusion domain, a transmembranedomain and a cytoplasmic domain; transfecting a nucleic acid sequenceencoding the protein portion into a eukaryotic host cell, wherein theeukaryotic host cell is not an insect eukaryotic host cell; andculturing the eukaryotic host cell to thereby make the protein thatstimulates a protective immune response in a subject.

In a further embodiment, the invention is a method of making a proteinthat stimulates a protective immune response in a subject, comprisingthe steps of separating a portion of a protein from a naturallyoccurring viral hemagglutinin to thereby form a protein portion, whereinthe protein portion includes at least a portion of a globular head, andat least a portion of at least one secondary structure that causes theglobular head to essentially retain its tertiary structure, and whereinthe protein portion lacks a membrane fusion domain, a transmembranedomain and a cytoplasmic domain; transfecting a nucleic acid sequenceencoding the protein portion into a eukaryotic host cell, wherein theeukaryotic host cell is not a stably transformed insect cell; andculturing the eukaryotic host cell to thereby make the protein thatstimulates a protective immune response in a subject.

In another embodiment, the invention is a method of making a proteinthat stimulates a protective immune response in a subject, comprisingthe steps of separating a portion of a protein from a naturallyoccurring viral hemagglutinin to thereby make a protein portion, whereinthe protein portion includes at least a portion of a globular head andat least a portion of at least one secondary structure that causes theglobular head to essentially retain its tertiary structure, and whereinthe protein portion lacks a membrane fusion domain, a transmembranedomain and a cytoplasmic domain; infecting a nucleic acid sequenceencoding the protein portion into an insect cell host cell (e.g., abaculovirus insect host cell, such as Sf9 or High5 cells); and culturingthe insect host cell to thereby make the protein that stimulates aprotective immune response in a subject.

The methods of making a protein that stimulates a protective immuneresponse in a subject can further include the step of deleting at leastone glycosylation site in the nucleic acid sequence encoding the proteinportion. The glycosylation site that is deleted can include anN-glycosylation site.

The eukaryotic host cells employed in the methods of the invention caninclude a Saccharomyces eukaryotic host cell, an insect eukaryotic hostcell (e.g., at least one member selected from the group consisting of aBaculovirus infected insect cell, such as Spodoptera frugiperda (Sf9) orTrichhoplusia ni (High5) cells; and a Drosophila insect cell, such asDme12 cells), a fungal eukaryotic host cell, a parasite eukaryotic hostcell (e.g., a Leishmania tarentolae eukaryotic host cell), CHO cells,yeast cells (e.g., Pichia) and a Kluyveromyces lactis host cell.

Suitable eukaryotic host cells and vectors can also include plant cells(e.g., tomato; chloroplast; mono- and dicotyledonous plant cells;Arabidopsis thaliana; Hordeum vulgare; Zea mays; potato, such as Solanumtuberosum; carrot, such as Daucus carota L.; and tobacco, such asNicotiana tabacum, Nicotiana benthamiana (Gils, M., et al., PlantBiotechnol J. 3:613-20 (2005); He, D. M., et al., Colloids Surf BBiointerfaces, (2006); Huang, Z., et al., Vaccine 19:2163-71 (2001);Khandelwal, A., et al., Virology. 308:207-15 (2003); Marquet-Blouin, E.,et al., Plant Mol Biol 51:459-69 (2003); Sudarshana, M. R., et al. PlantBiotechnol J. 4:551-9 (2006); Varsani, A., et al., Virus Res, 120:91-6(2006); Kamarajugadda S., et al., Expert Rev Vaccines 5:839-49 (2006);Koya V, et al., Infect Immun. 73:8266-74 (2005); Zhang, X., et al.,Plant Biotechnol J. 4:419-32 (2006)).

The proteins made by the methods of the invention and the compositionsof the invention can be purified and characterized employing well-knownmethods (e.g., gel chromatography, cation exchange chromatography,SDS-PAGE), as described herein.

For large scale production, fermentation techniques can be employed, asdescribed herein. Additional exemplary fermentation techniques caninclude a proposed cycle that can start with a culture inoculated into 6L of MRBR media, as described herein, held at about 30° C., about pH 7,and DO controlled to greater than about 30%. A 6 liter feed can then bestarted at least about 30 minutes after glucose exhaustion. The proposed6 liter feed media, when combined with 6 L of MRBR media, can providethe necessary conditions for E. coli growth based on about 52%utilization of carbon for growth. The feed may or may not include IPTG.The batch can be induced with at least 2 mM IPTG, introduced as a bolus,shortly after the feed is started to initiate production. The feed ratecan start at about 20 mL feed per hour per liter bioreactor volume andincrease over time based on the ability of the culture to accept moreglucose without glucose accumulation. The culture can be harvested whenthe feed is complete. The 6 liter feed media, about pH 6.0, can includeGlucose 180 g/L; KH₂PO₄ 2 g/L; NaH₂PO₄ (H₂O) 4 g/L; (NH₄)₂HPO₄ 12 g/L;(NH₄)₂HSO₄ 4 g/L; DL-Alanine 40 g/L; Citric Acid 4 g/L; MgSO₄(7H₂O) 5.5g/L; Trace Metals 6 mL; CaCl₂ 2.5 g/L; FeSO₄ 7H₂O 1 g/L.

Cell disruption and clarification in a large scale production caninclude removal of Triton X-100 from the resuspension buffer;dissolution of insolubles by the addition of 50 mM Tris, 25 mM NaCl, 8 Murea, about pH 8 to the lysate; addition of PEI (poly ethylamine) andsubsequent removal by centrifugation with one or more of the buffers toremove nucleic acids and/or aid in filtration; the addition offlocullants, such as Aerosil 380, Aerosil 200, Alkoxide Alu C, andCelpur; and subsequent removal by centrifugation to aid in filtration.Cation exchange chromatography can include the use of a process resin,adding a denaturing endotoxin removal step containing up to 8 M urea andup to about 2% Triton X-100, and a step gradient elution. The stepelution gradient can include about 100 to about 200 mM NaCl.

In yet another embodiment, the invention is a method of stimulatingprotective immunity in a subject, comprising the step of administeringto the subject a composition that includes a protein made by a methodcomprising the steps of separating a portion of a protein from anaturally occurring viral hemagglutinin to thereby form a proteinportion, wherein the protein portion includes at least a portion of aglobular head and at least a portion of at least one secondary structurethat causes the globular head to essentially retain its tertiarystructure, and wherein the protein portion lacks a membrane fusiondomain, a transmembrane domain and a cytoplasmic domain; transforming anucleic acid sequence encoding the portion into a prokaryotic host cell;and culturing the prokaryotic host cell to thereby make the protein thatstimulates protective immunity in a subject.

In still another embodiment, the invention is a method of stimulatingprotective immunity in a subject, comprising the step of administeringto the subject a composition that includes a protein portion of anaturally occurring viral hemagglutinin, wherein the protein portionincludes at least a portion of a globular head and at least a portion ofone secondary structure that causes the globular head to essentiallyretain its tertiary structure and wherein the protein portion lacks amembrane fusion domain, a transmembrane domain and a cytoplasmic domain.

In a further embodiment, the invention is a method of making a viralhemagglutinin protein that stimulates a protective immune response in asubject, comprising the steps of separating a portion of a protein froma naturally occurring viral hemagglutinin to thereby make a proteinportion, wherein the protein portion includes at least a portion of aglobular head and at least a portion of at least one secondary structurethat causes the globular head to essentially retain its tertiarystructure, and wherein the protein portion lacks a membrane fusiondomain, a transmembrane domain and a cytoplasmic domain; transfecting anucleic acid sequence encoding the portion in a eukaryotic host cell,wherein the eukaryotic host cell is not a Pichia pastoris eukaryotichost cell; and culturing the eukaryotic host cell to thereby make theprotein that stimulates a protective immune response in a subject.

In yet another embodiment, the invention is a method of making a viralhemagglutinin protein that stimulates a protective immune response in asubject, comprising the steps of separating a portion of a protein froma naturally occurring viral hemagglutinin to thereby make a proteinportion, wherein the protein portion includes at least a portion of aglobular head and at least a portion of at least one secondary structurethat causes the globular head to essentially retain its tertiarystructure, and wherein the protein portion lacks a membrane fusiondomain, a transmembrane domain and a cytoplasmic domain; transfecting anucleic acid sequence encoding the portion in a eukaryotic host cell,wherein the eukaryotic host cell is not a Drosophila melanogastereukaryotic host cell; and culturing the eukaryotic host cell to therebymake the protein that stimulates a protective immune response in asubject.

In still another embodiment, the invention is a method of making a viralhemagglutinin protein that stimulates a protective immune response in asubject, comprising the steps of transforming a prokaryotic host cellwith a nucleic acid sequence encoding at least one viral hemagglutininthat lacks a transmembrane domain and a cytoplasmic domain; andculturing the prokaryotic cell to thereby make the protein thatstimulates a protective immune response in a subject.

An additional embodiment of the invention is a method of stimulatingprotective immunity in a subject, comprising the step of administeringto the subject a composition that includes a protein made by a methodcomprising the steps of transforming a prokaryotic host cell with anucleic acid sequence encoding at least one viral hemagglutinin thatlacks a transmembrane domain and a cytoplasmic domain; and culturing theprokaryotic host cell to thereby make the protein that stimulates aprotective immune response in a subject.

In another embodiment, the invention is a method of stimulatingprotective immunity in a subject, comprising the step of administeringto the subject a composition that includes a protein having at least oneviral hemagglutinin that lacks a transmembrane domain and a cytoplasmicdomain, wherein the protein was expressed in a prokaryotic cell.

In still another embodiment, the invention is a composition comprisingat least a portion of at least one pathogen-associated molecular patternand a protein portion of a naturally occurring viral hemagglutinin,wherein the protein portion of the naturally occurring viralhemagglutinin includes at least a portion of a globular head and atleast a portion of at least one secondary structure that causes theglobular head to essentially retain its tertiary structure, and whereinthe protein portion of the naturally occurring viral hemagglutinin lacksa membrane fusion domain, a transmembrane domain, and a cytoplasmicdomain.

Pathogen-associated molecular patterns (PAMPs), such as a flagellin or abacterial lipoprotein, refer to a class of molecules (e.g., protein,peptide, carbohydrate, lipid, lipopeptide, nucleic acid) found inmicroorganisms that, when bound to a pattern recognition receptor (PRR),can trigger an innate immune response. The PRR can be a Toll-likeReceptor (TLR).

TLRs are the best characterized type of Pattern Recognition Receptor(PRR) expressed on antigen-presenting cells (APC). APC utilize TLRs tosurvey the microenvironment and detect signals of pathogenic infectionby engaging the cognate ligands of TLRs, PAMPs. PAMP and TLR interactiontriggers the innate immune response, the first line of defense againstpathogenic insult, manifested as release of cytokines, chemokines andother inflammatory mediators; recruitment of phagocytic cells; andimportant cellular mechanisms which lead to the expression ofcostimulatory molecules and efficient processing and presentation ofantigens to T-cells. TLRs control both innate and the adaptive immuneresponses.

The binding of PAMPs to TLRs activates innate immune pathways. Targetcells can result in the display of co-stimulatory molecules on the cellsurface, as well as antigenic peptide in the context of majorhistocompatibility complex molecules (see FIG. 40). The compositions,fusion proteins or polypeptides of the invention can include a PAMP(e.g., a flagellin) that binds to a TLR (e.g., TLR5), promotingdifferentiation and maturation of the APC, including production anddisplay of co-stimulatory signals (see FIG. 40). The compositions can beinternalized by its interaction with the TLR and processed through thelysosomal pathway to generate antigenic peptides, which are displayed onthe surface in the context of the major histocompatibility complex.

The compositions, fusion proteins and polypeptides of the inventionemploy pathogen-associated molecular patterns (TLR agonists) thattrigger cellular events resulting in the expression of costimulatorymolecules, secretion of critical cytokines and chemokines; and efficientprocessing and presentation of antigens to T-cells. As discussed above,TLRs recognize PAMPs including bacterial cell wall components (e.g.,bacterial lipoproteins and lipopolysaccharides), bacterial DNA sequencesthat contain unmethylated CpG residues and bacterial flagellin. TLRs actas initiators of the innate immune response and gatekeepers of theadaptive immune response (Medzhitov, R., et al., Cold Springs Harb.Symp. Quant. Biol. 64:429 (1999); Pasare, C., et al., Semin, Immunol16:23 (2004); Medzhitov, R., et al., Nature 388:394 (1997); Barton, G.M., et al., Curr. Opin. Immunol 14:380 (2002); Bendelac, A., et al., J.Exp. Med. 195:F19 (2002)).

As discussed above, the binding of PAMPs to TLRs activates immunepathways for use in the compositions, fusion proteins and polypeptidesof the invention, which can be employed in stimulating the immune systemin a subject. The compositions, fusion proteins and polypeptides of theinvention can trigger an immune response to an antigen (e.g., a viralprotein, such as an influenza viral) and trigger signal transductionpathways of the innate and adaptive immune system of the subject tothereby stimulate the immune system of a subject. Stimulation of theimmune system of the subject may prevent infection by an antigen or avirus (e.g., an influenza virus) and thereby treat the subject orprevent the subject from disease, illness and, possibly, death.

In an additional embodiment, the invention is a composition comprising aflagellin component that is at least a portion of a flagellin, whereinthe flagellin component includes at least one cysteine residue andwhereby the flagellin component activates a Toll-like Receptor 5.

“Flagellin component,” as used herein, means at least part of or theentirety of a flagellin.

“Activates,” when referring to a Toll-like Receptor (TLR), means thatthe component (e.g., a flagellin component or a Toll-like Receptoragonist component) stimulates a response associated with a TLR. Forexample, bacterial flagellin activates TLR5 and host inflammatoryresponses (Smith, K. D., et al., Nature Immunology 4:1247-1253 (2003)).Bacterial lipopeptide activates TLR1; Pam3Cys, Pam2Cys activate TLR2;dsRNA activates TLR3; LBS (LPS-binding protein) and LPS(lipopolysaccharide) activate TLR4; imidazoquinolines (anti-viralcompounds and ssRNA) activate TLR7; and bacterial DNA (CpG DNA)activates TLR9. TLR1 and TLR6 require heterodimerization with TLR2 torecognize ligands (e.g., TLR agonists, TLR antagonists). TLR1/2 areactivated by triacyl lipoprotein (or a lipopeptide, such as Pam3Cys),whereas TLR6/2 are activated by diacyl lipoproteins (e.g., Pam2Cys),although there may be some cross-recognition. In addition to the naturalligands, synthetic small molecules including the imidazoquinolines, withsubclasses that are specific for TLR7 or TLR8 can activate both TLR7 andTLR8. There are also synthetic analogs of LPS that activate TLR4, suchas monophosphoryl lipid A [MPL].

TLR activation can result in signaling through MyD88 and NF-κB. There issome evidence that different TLRs induce different immune outcomes. Forexample, Hirschfeld, et al. Infect Immun 69:1477-1482 (2001)) and Re, etal. J Biol Chem 276:37692-37699 (2001) demonstrated that TLR2 and TLR4activate different gene expression patterns in dendritic cells.Pulendran, et at J Immunol 167:5067-5076 (2001)) demonstrated that thesedivergent gene expression patterns were recapitulated at the proteinlevel in an antigen-specific response, when lipopolysaccharides thatsignal through TLR2 or TLR4 were used to guide the response (TLR4favored a Th1-like response with abundant IFNγ secretion, while TLR2favored a Th2-line response with abundant IL-5, IL-10, and IL-13 withlower IFNγ levels). There is redundancy in the outcome of signalingthrough different TLRs.

Activation of TLRs can result in increased effector cell activity thatcan be detected, for example, by measuring IFNγ-secreting CD8+ cells(e.g., cytotoxic T-cell activity); increased antibody responses that canbe detected by, for example, ELISA, virus neutralization, and flowcytometry (Schnare, M., et al., Nat Immunol 2:947 (2001); Alexopoulou,L., et al., Nat Med 8:878 (2002); Pasare, C., et al., Science 299:1033(2003); Napolitani, G., et al., Nat Immunol 6:769 (2005); andApplequist, S. E., et al., J Immunol 175:3882 (2005)).

The composition comprising a flagellin component that is at least aportion of a flagellin, wherein the flagellin component includes atleast one cysteine residue and whereby the flagellin component activatesa Toll-like Receptor 5 can further include at least a portion of atleast one member selected from the group consisting of a Toll-likeReceptor 1 agonist, a Toll-like Receptor 2 agonist (e.g., Pam3Cys,Pam2Cys), a Toll-like Receptor 3 agonist, a Toll-like Receptor 4agonist, a Toll-like Receptor 6 agonist, a Toll-like Receptor 7 agonist,a Toll-like Receptor 8 agonist a Toll-like Receptor 9 agonist, aToll-like Receptor 10 agonist, a Toll-like Receptor 11 agonist and aToll-like Receptor 12 agonist.

The a Toll-like Receptor 1 agonist, a Toll-like Receptor 2 agonist, aToll-like Receptor 3 agonist, a Toll-like Receptor 4 agonist, aToll-like Receptor 6 agonist, a Toll-like Receptor 7 agonist, aToll-like Receptor 8 agonist a Toll-like Receptor 9 agonist a Toll-likeReceptor 10 agonist, a Toll-like Receptor 11 agonist and a Toll-likeReceptor 12 agonist employed in the compositions, such as compositionscomprising a flagellin component that is at least a portion of aflagellin, wherein the flagellin component includes at least onecysteine residue and whereby the flagellin component activates TLR5, canfurther include at least one additional cysteine residue.

In one embodiment, at least one cysteine residue substitutes for atleast one amino acid residue in a naturally occurring flagellin aminoacid sequence of the flagellin component.

The cysteine residue that substitutes for at least one amino acidresidue in a naturally occurring flagellin amino acid sequence of theflagellin component can be remote to at least one amino acid of theToll-like Receptor 5 recognition site of the flagellin component.“Toll-like Receptor 5 recognition site,” means that part of the TLR5ligand (e.g., TLR5 agonist) that interacts with TLR5 to mediate acellular response. “Toll-like Receptor 5 recognition site” is alsoreferred to as a “Toll-like Receptor 5 activation site” and a “Toll-likeReceptor 5 activation domain.”

Likewise, “Toll-like Receptor recognition site,” means that part of theToll-like Receptor ligand (e.g., a Toll-like Receptor agonist) thatinteracts with its respective TLR to mediate a cellular response.“Toll-like Receptor recognition site” is also referred to as a“Toll-like Receptor activation site” and a “Toll-like Receptoractivation domain.”

The cysteine residue that substitutes for at least one amino acidresidue in a naturally occurring flagellin amino acid sequence of theflagellin component can also be remote to at least one amino acid of theflagellin component involved in binding to the toll like receptor 5.

In another embodiment, the flagellin component includes at least aportion of a naturally occurring flagellin amino acid sequence incombination with the cysteine residue.

The cysteine residue used in combination (also referred to herein as“added cysteine”) with at least a portion of a naturally occurringflagellin amino acid sequence can be at least one member selected fromthe group consisting of the amino-terminal amino acid of the flagellincomponent or the Toll-like Receptor agonist component and thecarboxy-terminal amino acid of the flagellin component or the Toll-likeReceptor agonist component. The cysteine residue used in combinationwith at least a portion of a naturally occurring flagellin amino acidsequence can be remote to at least one amino acid of the Toll-likeReceptor 5 recognition site of the flagellin component or remote to atleast one amino acid of the Toll-like Receptor recognition site of theToll-like Receptor agonist component.

The cysteine residue used in combination with at least a portion of anaturally occurring flagellin amino acid sequence can be remote to atleast one amino acid of the flagellin component involved in binding tothe Toll-like Receptor 5 or remote to at least one amino acid of aToll-like Receptor agonist component involved in binding to theToll-like Receptor.

In another embodiment, the flagellin component lacks at least a portionof a hinge region of a naturally occurring flagellin amino acidsequence.

The composition comprising a flagellin component that is at least aportion of a flagellin, wherein the flagellin component includes atleast one cysteine residue and whereby the flagellin component activatesa Toll-like Receptor 5 can further include at least a portion of atleast one antigen (e.g., an influenza antigen, such as an influenza A, Bor C antigen).

The antigen can be an essentially hydrophobic antigen, such as amaturational cleavage site antigen of HA. The maturational cleavage siteantigen can be at least one member selected from the group consisting of(SEQ ID NOS: 530, 532, 533, 534, 535, 600, 601, 602, 603 andNVPEKQTRGIFGAIAGFIE (H3) (SEQ ID NO: 796), NIPSIQSRGLFGAIAGFIE (H1) (SEQID NO: 797), PAKLLKERGFFGAIAGFLE (FLU B) (SEQ ID NO: 798),RERRRKKRGLFGAIAGFIE (H5) (SEQ ID NO: 799), RGLXGAIAGFIE (SEQ ID NO:821), RGLXGAIAGFIE (SEQ ID NO: 822).

“Essentially hydrophobic,” as used herein, means that the antigen haslimited solubility in an aqueous solution or environment. Thehydrophobic nature of peptides or proteins can be determined andcompared, for example, by the Kyte-Doolitle hydrophobicity scale (Kyte,J., et al., Mol. Biol. 157: 105-132 (1982), (1982)), which assigns anumerical value for each of the 20 amino acids according to relativehydrophobicity. A positive value indicates a hydrophobic amino acid. Anegative value indicates a hydrophilic amino acid. The average of thesevalues for an individual peptide or polypeptide (calculated by addingthe individual hydrophobicity values for each amino acid of thepolypeptide or protein and dividing the total value by the number ofamino acids in the polypeptide or protein) provides an index of overallhydrophobicity known as the “grand average of hydropathicity,” alsoreferred to as “GRAVY.” A GRAVY value greater than zero indicates theprotein or peptide is essentially hydrophobic. A GRAVY value less thanzero indicates the protein or peptide is essentially hydrophilic. Theindividual hydrophobicity values, for the 20 naturally occurring aminoacids according to the Kyte-Doolittle scale (Kyte, J., et al., Mol.Biol. 157: 105-132 (1982)), are as follows:

Alanine 1.8 Arginine −4.5 Asparagine −3.5 Aspartic acid −3.5 Cysteine2.5 Glutamine −3.5 Glutamic acid −3.5 Glycine −0.4 Histidine −3.2Isoleucine 4.5 Leucine 3.8 Lysine −3.9 Methionine 1.9 Phenylalanine 2.8Proline −1.6 Serine −0.8 Threonine −0.7 Tryptophan −0.9 Tyrosine −1.3Valine 4.2

The hydrophobicity of antigens, such as protein or peptide antigens, foruse in the compositions and methods of the invention can be determinedby calculating a GRAVY score based on the hydrophobicity values of theabove Kyte-Doolittle scale. For example, GRAVY scores, which indicateessentially hydrophobic peptides, were calculated by addition of thehydrophobicity values (supra) of individual amino acids for thefollowing maturational cleavage site amino peptides:

Maturational Cleavage Site GRAVYNVPEKQTRGIFGAIAGFIE (A/H3N2) (SEQ ID NO: 800) 0.053NVPQIESRGLFGAIAGFIE (A/H2N1) (SEQ ID NO: 801) 0.453NIPSIQSRGLFGAIAGFIE (A/H1N1) (SEQ ID NO: 802) 0.611RGLFGAIAGFIE (Influenza A conserved region) (SEQ ID NO: 803) 1.067PAKLLKERGFFGAIAGFLE (Influenza B) (SEQ ID NO: 804) 0.400RGFFGAIAGFLE (Influenza B conserved region) (SEQ ID NO: 805) 0.925

Likewise, GRAVY scores, which indicate essentially hydrophilic peptides,were calculated for the following peptides:

Amino Acid Sequence GRAVY human influenza M2e (SEQ ID NO: 806) −1.129SLLTEVETPIRNEWGSRSNDSSDP Vietnam influenza M2e (SEQ ID NO: 807) −0.907GSGAGSLLTEVETPTRNEWECRCSDSSDP Hong Kong influenza M2e (SEQ ID NO: 808)−0.507 GSGAGSLLTEVETLTRNGWGCRCSDSSDP West Nile Virus E peptide 001−0.475 (SEQ ID NO: 809) LTSGHLKCRVKMEKLQLKGTDengue 2 E peptide (SEQ ID NO: 810) −0.333 EAEPPFGDSYIIIGVEPGQLKLNWFKKBCRABL wt peptide (SEQ ID NO: 811) −1.129 SLLTEVETPIRNEWGSRSNDSSDP

An antigen can be any molecule (e.g., protein, peptide, glycoprotein,glycopeptide, carbohydrate, lipid, lipopeptide, polysaccharide) that canbe recognized by the components of the immune system regardless ofwhether it can trigger activation of the immune system. The antigen canbe a fragment or portion of a naturally occurring antigen or a syntheticmolecule that mimics the naturally occurring antigen or a portion of thenaturally occurring antigen.

The antigen can be a viral antigen. A “viral antigen,” as used herein,refers to any portion of a virus (e.g., influenza virus, flavivirus)that generates an immune response in a subject either when employed incombination with a TLR agonist (e.g., a flagellin, Pam2Cys, Pam3Cys) orin the absence of a TLR agonist. The viral antigen can be a portion or afragment of a naturally occurring virus or a synthetic molecule thatmimics a naturally occurring virus, such as a recombinant or syntheticprotein (e.g., influenza virus, flavivirus), peptide, lipid,carbohydrate, that generates an immune response in the subject. Theinfluenza antigen can include at least one member selected from thegroup consisting of an influenza A antigen, influenza B antigen and aninfluenza C antigen. The influenza antigen can be an influenza virusintegral membrane protein, such as HA, or a protein portion of HA, suchas HA1-1 and HA1-2.

The antigen can be at least a portion of an influenza Matrix 2 (M2)protein, including at least a portion of the ectodomain of an influenzaM2 protein (M2e).

Matrix protein 2 (M2 or M2 protein) is a proton-selective integralmembrane ion channel protein of the influenza A virus. M2 is abundantlyexpressed at the plasma membrane of virus-infected cells, but isgenerally underexpressed by virions. For example, a portion of an M2sequence of influenza A is SEQ ID NO: 508, which is encoded by SEQ IDNO: 509. The native form of the M2 protein is a homotetramer (i.e., fouridentical disulfide-linked M2 protein molecules). Each of the units arehelices stabilized by two disulfide bonds. M2 is activated by low pH.Each of the M2 protein molecules in the homotetramer consists of threedomains: a 24 amino acid outer or N (amino)-terminal domain (e.g., SEQID NO: 510; also referred to herein as a “human consensus sequence”),which is encoded by SEQ ID NO: 511; a 19 hydrophobic amino acidtransmembrane region, and a 54 amino acid inner or C (carboxy)-terminaldomain. The M2 protein can vary depending upon the influenza viralsubtype (e.g., H1 and H5 subtypes of influenza A) and influenza viralsource (e.g., Puerto Rico, Thailand, New York, Hong Kong), as shown, forexample, in exemplary amino-terminal sequences of M2 proteins (SEQ IDNOS: 544-556 and 570-578) and as described in PCT/US2005/046662(WO2006/069262).

The M2 protein has an important role in the life cycle of the influenzaA virus. It is important in the uncoating stage where it permits theentry of protons into the viral particle, which lowers the pH inside thevirus, resulting in dissociation of the viral matrix protein M1 from theribonucleoprotein RNP. As a consequence, the virus coat is removed andthe contents of the virus are released from the endosome into thecytoplasm of the host cell for infection.

The function of the M2 channel can be inhibited by antiviral drugs, suchas amantadine and rimantadine, which prevent the virus from infectingthe host cell. Such antiviral drugs usually bind the transmembraneregion of the M2 protein and sterically block the ion channel created bythe M2 protein, which prevents protons from entering and uncoating thevirion.

The M2 protein for use in the compositions and methods of the inventioncan that include at least a portion of SEQ ID NO: 510 encoded by SEQ IDNO: 511 or at least a portion of SEQ ID NO: 544, encoded by SEQ ID NO:603. The M2 protein can further include at least one member selectedfrom the group consisting of SEQ ID NO: 512, SEQ ID NO: 516, SEQ ID NO:531; SEQ ID NO: 536 (Flu A H5N1 M2e, 2004 Viet Nam Isolate with serinereplacing cysteine); SEQ ID NO: 537 (Flu A H5N1 M2e, 2004 Viet NamIsolate); SEQ ID NO: 538 (Flu A H5N1 M2e, Hong Kong 97 Isolate withserine replacing cysteine); SEQ ID NO: 539 (Flu A H5N1 M2e, Hong Kong 97Isolate); SEQ ID NO: 540 (Flu A H7N2 M2e Chicken/New York 95 Isolatewith serine replacing cysteine); SEQ ID NO: 541 (Flu A H7N2 M2e,Chicken/New York 95 Isolate); SEQ ID NO: 542 (Flu A H9N2 M2e, Hong Kong99 Isolate with serine replacing cysteine); and SEQ ID NO: 543 (Flu A,Hong Kong 99 Isolate). Certain cysteine residues, for example, aminoacids 16 and 18 of SEQ ID NO: 537; amino acids 17 and 19 of SEQ ID NOS:539, 541 and 543 in the naturally occurring sequence of at least aportion of M2 protein are replaced with a serine (see, SEQ ID NOS: 538,540, 542 and 544, respectively).

The compositions comprising a flagellin component that is at least aportion of a flagellin, wherein the flagellin component includes atleast one cysteine residue and whereby the flagellin component activatesa Toll-like Receptor 5 that further includes an antigen (e.g., amaturational cleavage site peptide, protein portion of HA, such asHA1-1, HA1-2) can be co-administered or admixed with another flagellincomponent that is at least a portion of a flagellin, wherein theflagellin component includes at least one cysteine residue and wherebythe flagellin component activates a Toll-like Receptor 5 that furtherincludes a different, distinct antigen (e.g., M2e) or a fusion proteinof a TLR agonist and an antigen (e.g., STF2.HA1-1, STF2.HA1-2,STF2Δ.HA1-1, STF2Δ.HA1-2, STF2.M2e, STF2.4×M2e, STF2Δ.M2e, STF2Δ.4×M2e).

The antigen included in the compositions and employed in the methods ofthe invention can be at least a portion of a microbial-related antigen,for example, antigens of pathogens, such as viruses, fungi or bacteria.Antigens can also be microorganism-related, and other disease-relatedantigens, such as antigens associated with allergies and cancer. Theantigen can also be at least a portion of an antigen of a bacteria, avirus, a fungi, a yeast, a protozoa, a metazoa, a tumor, a malignantcell, a plant cell, an animal cell, a hormone and an amyloid-β peptide.The antigen can be a peptide, a polypeptide, a lipoprotein, aglycoprotein and a mucoprotein.

The antigen included in the compositions and employed in the methods ofthe invention can be a pathogen-related antigen. “Pathogen-relatedantigen,” as used herein, refers to any molecule (e.g., protein,peptide, carbohydrate, lipoprotein, polysaccharide) that is associatedwith a pathogen. Exemplary pathogen-related antigens include, forexample, antigens of vaccinia, avipox virus, turkey influenza virus,bovine leukemia virus, feline leukemia virus, avian influenza, chickenpneumovirosis virus, canine parvovirus, equine influenza, FHV, NewcastleDisease Virus (NDV), Chicken/Pennsylvania/1/83 influenza virus,infectious bronchitis virus; Dengue virus, measles virus, Rubella virus,pseudorabies, Epstein-Barr Virus, HIV, SIV, EHV, BHV, HCMV, Hantaan, C.tetani, mumps, Morbillivirus, Herpes Simplex Virus type 1, HerpesSimplex Virus type 2, Human cytomegalovirus, Hepatitis A Virus,Hepatitis B Virus, Hepatitis C Virus, Hepatitis E Virus, RespiratorySyncytial Virus, Human Papilloma Virus, Influenza Virus, Salmonella,Neisseria, Borrelia, Chlamydia, Bordetella, Plasmodium (e.g., Plasmodiumfalciparum and Plasmodium vivax), Toxoplasma, Cryptococcus,Streptococcus, Staphylococcus, Haemophilus, Diptheria, Tetanus,Pertussis, Escherichia, Candida, Aspergillus, Entamoeba, Giardia andTrypanasoma.

The antigen included in the compositions and employed in the methods ofthe invention can include bacterial capsular antigens. Exemplarybacterial capsular antigens include, for example, at least one memberselected from the group consisting of a Group B Streptococcus, includingStreptococcus agalactiae, capsular polysaccharides type 1a, 1b, Ia/c,II, III, IV, V and VI; Streptococcus pneumoniae capsular polysaccharidestypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F,18C, 19A, 20, 22F, 23F, and 33F or any combination of these (typingaccording to the Danish system); Stapylococcus aureus capsularpolysaccharides type 5, 8 and 336; Bacillus anthracis vegetative statecapsular poly gamma D glutamic acid; Mycobacteria tuberculosislipo-arabino mannan capsule; Plasmodium flaciparum surfaceoligosaccharide; Neisseria meningitidis capsular polysaccharides ofgroup A, B, C, X, Y and W135 or any combination thereof.

Compositions comprising the flagellin component that is at least aportion of a flagellin, wherein the flagellin component includes atleast one cysteine residue and whereby the flagellin component activatesa Toll-like Receptor 5 can include an antigen that is produced bychemical modification of the side-chains of the antigen (e.g., capsularantigens, pathogen-related antigens, influenza antigens, flavivirusantigens) in a manner that generates an active group that is coupled(also referred to herein as chemical conjugation) to a compatiblereactive group created on the substituted flagellins of the invention(see infra). Derivatization (modification) of the capsular material iscarried out so that a small minority of the side-chains are modified(about 5%) to avoid destruction of the majority of immunoreactivestructures on the capsular material.

The antigen included in the compositions and employed in the methods ofthe invention can be at least a portion of at least one member selectedfrom the group consisting of a West Nile viral protein, a Langat viralprotein, a Kunjin viral protein, a Murray Valley encephalitis viralprotein, a Japanese encephalitis viral protein, a Tick-borneencephalitis viral protein, Dengue 1 viral protein, Dengue 2 viralprotein, Dengue 3 viral protein, Dengue 4 viral protein, hepatitis Cviral protein and a Yellow fever viral protein (see, for example,PCT/US2006/001623 (WO2006/078657)).

The genus flavivirus is in the virus family Flaviviridae and consists ofabout 70 viruses. Mosquito or ticks transmit most of these viruses.Several flaviviruses are significant human pathogens, including the fourdengue viruses (Den1, Den2, Den3 and Den4), yellow fever (YF), Japaneseencephalitis (JE), West Nile (WN, also referred to herein as “WNV”) andTick-borne encephalitis (TBE) (Weaver S. C., et al., Nat Rev Microbiol10: 789-801 (2004)). The flavivirus genus is divided into a number ofserogroups based on cross-neutralization tests, including the dengueserogroup that contains four serologically and genetically distinctviruses termed DEN-1, DEN-2, DEN-3 and DEN-4.

Flaviviruses are small, enveloped viruses with icosahedral capsids. Theflavivirus genome is a single-stranded positive-sense RNA (about 11 kb)that is directly translated by the host cell machinery followinginfection. The viral genome is translated as a single polypeptide thatundergoes co- and post-translational cleavage by viral and cellularenzymes to generate three structural proteins of the flavivirus (thecapsid (C), the membrane (M) and the envelope (E) proteins); and sevennonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5)(Weaver, et al., Annu Rev Microbiol 1990:44-649 (2004)). The viralcapsid is composed of the C-protein, while both the M- and envelopeproteins are located on the envelope surface of the virion (Weaver, S.C., et al., Nat. Rev. Microbiol. 10:789-801 (2004); Chambers et al.,Annu Rev. Microbiol. 44: 649-688 (1990)). A major immunogen forflaviviruses is the membrane envelope protein.

A flavivirus can enter a host cell when the viral envelope protein bindsto a receptor and responds by conformational rearrangement to thereduced pH of an endosome. The conformational change induces fusion ofviral and host-cell membranes.

The envelope of a flavivirus may function as a receptor binding proteinand to facilitate fusion of the virus and host cell membrane. Envelopeproteins of flaviviruses have common structural (domains I, II and III)and functional features (receptor binding of virus and host cell andfusion functions) and are class II fusion glycoproteins (Lescar et al.,Cell 105:137-148 (2001)).

In the pre-fusion conformation, envelope proteins form homodimers on theouter surface of the virus particles (Rey, et al., Nature 375:291-298);Kuhn, et al., Cell 108:717-725 (2002); Mukhopadhyay, et al., Science302:248 (2003)). Each envelope protein monomer folds into threestructural domains (domains I, II and III) predominantly composed ofβ-strands. Domain I (also referred to herein as “I” or “DI”) iscentrally located in the structure and has an N-glycosylation site inglycosylated envelope proteins. Domain II (also referred to herein as“II” or “DII”) of the envelope protein promotes dimerization and has afusion loop that inserts into the target host membrane during thepH-dependent fusion of the virus (Modis, et al., Nature 427:313-319(2004); Bressanelli, et al., EMBO J 23:728-738 (2004)). Domain III (alsoreferred to herein as “III” or “DIII”) is at the carboxy-terminus of theenvelope protein. Domain III is also referred to as “domain B” inearlier antigenic mapping studies. Domain III has several epitopes thatcan elicit virus-neutralizing antibodies (Roehrig, Adv Virus Res59:141-175 (2003)).

Domain I of the Tick-borne encephalitis envelope protein corresponds toamino acids 1-51, 137-189 and 285-302 of SEQ ID NO: 777; domain II ofthe Tick-borne encephalitis envelope protein of SEQ ID NO: 777corresponds to amino acids 52-136 and 190-284; and domain IIIcorresponds to amino acids 303-395 of SEQ ID NO: 777. (Rey, F. A., etal., Nature 375:291-298 (1995)). SEQ ID NO: 777 is encoded by SEQ ID NO:778. Domain I of the Dengue 2 flavivirus envelope protein corresponds toamino acids 1-52, 132-193 and 280-296 of SEQ ID NO: 763; domain IIcorresponds to amino acids 53-131 and 194-279 of SEQ ID NO: 763; anddomain III corresponds to amino acids 297-495 of SEQ ID NO: 763 (Modis,Y., et al., Nature 427:313-319 (2004)). The location of domains I, IIand III of other flavivirus (e.g., West Nile virus, Japaneseencephalitis, Dengue 1 virus, Dengue 3 virus and Dengue 4 virus) isbased on homology of the Tick-borne encephalitis envelope proteindomains and the Dengue 2 envelope protein domains. Thus, referenceherein to domains of flavivirus proteins, in particular, flavivirusesother than Tick-borne encephalitis flavivirus envelope proteins andDengue 2 flavivirus envelope proteins, are based on homology to domainsin the Tick-borne encephalitis flavivirus envelope protein and theDengue 2 flavivirus envelope protein.

The domain III of the envelope protein of the DEN flavivirus encodes themajority of the flavivirus type-specific contiguous critical/dominantneutralizing epitopes (Roehring, J. T., Adv. Virus Res. 59:141 (2003)),including the four DEN (DEN1, DEN2, DEN3, DEN4) viruses. Flavivirusenvelope proteins are highly homologous. Exemplary envelope proteinsequences are SEQ ID NOS: 642, 763, 765, 767, 769 and 774.

West Nile virus (WNV) is a single-stranded positive sense RNA envelopevirus. It was first isolated and identified in the West Nile region ofUganda in 1937 from a febrile female adult (Smithburn, et al., Am J TropMed Hyg 3:9-18 (1954)).

Japanese encephalitis (JE) virus is localized in Asia and northernAustralia (about 50,000 cases with about 10,000 deaths annually).

The Dengue (DEN) disease is caused by four mosquito-borne, serologicallyrelated flaviviruses known as DEN-1 (also referred to herein as “Den1”or Den 1″), DEN-2 (also referred to herein as “Den2” or “Den 2”), DEN-3(also referred to herein as “Den3” or “Den 3”), and DEN-4 (also referredto herein as “Den4” or Den 4″). The compositions, fusion proteins andpolypeptides of the invention can include Den 1 SEQ ID NO.: 623; Den 1PR 94 (Puerto Rico, 1994) SEQ ID NO: 624; Den 3 SEQ ID NO: 626; and Den4 SEQ ID NO: 627. SEQ ID NOS: 623, 624, 625, 626 and 627 are portions ofdomain III of Den1, Den2, Den3 and Den4 flaviviruses.

“EI,” “EII,” and “EIII,” as used herein, refer to domains I, II and III,respectively, of the West Nile flavivirus envelope protein. “JEI,”“JEII,” and “JEIII,” as used herein, refer to domains I, II and III,respectively, of the Japanese encephalitis flavivirus envelope protein.“Den1 I,” “Den1 II,” and “Den1 III,” as used herein refer to domains I,II and III, respectively, of the Dengue 1 flavivirus envelope protein.Likewise, designations for the domains of envelope proteins of otherflaviviruses are referenced by the flavivirus name followed by thedomain number (e.g., (Tick-borne) TBI (Tick-borne), TBII, TBIII, Den2 I,Den2 II, Den2 III).

The portion of an envelope protein of a flavivirus can include at leastone member selected from the group consisting of at least a portion ofdomain I, at least a portion of domain II and at least a portion ofdomain III. When a domain is designated with a “+,” for example “EIII+”or “JEIII+,” the portion of the envelope protein referenced as “III” isone component of the total of that domain plus at least one of at leasta portion of either or both of domains I and II. For example, “EIII+,”as used herein, means the compositions, fusion proteins and polypeptidesof the invention include domain III and at least a portion of domain I.“EIII+” is also referred to as “EI/III.” “JEIII+” is also referred to as“JEI/III.” Similarly, when compositions include domains of envelopeproteins of flavivirus, the domains can be any combination of domains I,II, and III and can be designated based on the domain. For example,EI/II includes domain I and II of the West Nile flavivirus. The absenceof a “+” in reference to a domain (e.g., EIII, JEIII, Den1 III) of anenvelope protein employed in the compositions, fusion proteins andpolypeptides of the invention means that the composition, fusion proteinand polypeptide includes the referenced domain. For example, “Den1 III”means the compositions, fusion proteins and compositions include domainIII, not domains I and II, of the Dengue 1 virus.

The West Nile viral envelope protein can include at least a portion ofat least one member selected from the group consisting of SEQ ID NO:610, which is an EIII+ amino acid sequence, the italicized amino acidsare domain I of the envelope protein and the remaining sequence isdomain III of the envelope protein; SEQ ID NO: 611, West Nile virus,Stanford, Conn., also referred to as “West Nile S”; SEQ ID NO: 612, WestNile virus, New York, N.Y., also referred to as “West Nile NY”; and SEQID NO: 613, SEQ ID NO: 610 is encoded by SEQ ID NO: 614.

The Langat virus envelope protein for use in the compositions, fusionproteins and polypeptides of the invention can include at least aportion of SEQ ID NO: 615. The Kunjin virus envelope protein can includeat least a portion of SEQ ID NO: 616. The Murray Valley encephalitisenvelope protein can include at least a portion of SEQ ID NO: 617. TheJapanese encephalitis envelope protein can include at least one memberselected from the group consisting of at least a portion of SEQ ID NO:618 and SEQ ID NO: 619. The Tick-borne encephalitis envelope protein caninclude at least a portion of SEQ ID NO: 620. The Yellow fever virusenvelope protein can include at least a portion of SEQ ID NO: 621. Theenvelope protein of a flavivirus can include at least a portion of atleast one member selected from the group consisting of SEQ ID NO: 622and SEQ ID NO: 643. SEQ ID NOS: 615, 616, 617, 618, 619, 620, 621, 622and 643 are portions of domain III of the viral envelope protein.

The antigen can be chemically conjugated to flagellin components andToll-like Receptor agonist components. Chemical conjugation (alsoreferred to herein as “chemical coupling”) can include conjugation by areactive group, such as a thiol group (e.g., a cysteine residue) or byderivatization of a primary (e.g., a amino-terminal) or secondary (e.g.,lysine) group. Different crosslinkers can be used to chemicallyconjugate TLR ligands (e.g., TLR agonists) to proteins (e.g., antigens,compositions of the invention, HA and M2e constructs of the invention)or other molecules (e.g., nucleic acids, polysaccharides). Exemplarycross linking agents are commerically available, for example, fromPierce (Rockland, Ill.). Methods to chemically conjugate the antigen tothe flagellin component are well-known and include the use ofcommercially available cross-linkers, such as those described herein.

For example, conjugation of peptide or protein antigens to a flagellincomponent or a Toll-like Receptor agonist component of the invention canbe through at least one cysteine residue of the flagellin component orthe Toll-like Receptor component and at least one cysteine residue of aprotein (e.g., an influenza antigen, such as HA, M2e) employingestablished techniques. The protein can be derivatized with ahomobifunctional, sulfhydryl-specific crosslinker; the protein is thendesalted to remove the unreacted crosslinker; and then the peptide orprotein partner added and conjugated via at least one cysteine residuecysteine. Exemplary reagents for use in the conjugation methods can bepurchased commercially from Pierce (Rockland, Ill.), for example, BMB(Catalog No: 22331), BMDB (Catalog No: 22332), BMH (Catalog No: 22330),BMOE (Catalog No: 22323), BM[PEO]₃ (Catalog No: 22336), BM[PEO]₄(Catalog No: 22337), DPDPB (Catalog No: 21702), DTME (Catalog No:22335), HBVS (Catalog No: 22334).

Alternatively, cysteine-containing proteins and antigens can also beconjugated to lysine residues on flagellin components, flagellin,Toll-like Receptor agonist components and Toll-like Receptor agonists ofthe invention. A protein containing no cysteine residues is derivatizedwith a heterobifunctional amine and sulfhydryl-specific crosslinker.After desalting, the cysteine-containing partner is added andconjugated. Exemplary reagents for use in the conjugation methods can bepurchased from Pierce (Rockland, Ill.), for example, AMAS (Catalog No:22295), BMPA (Catalog No. 22296), BMPS (Catalog No: 22298), EMCA(Catalog No: 22306), EMCS (Catalog No: 22308), GMBS (Catalog No: 22309),KMUA (Catalog No: 22211), LC-SMCC (Catalog No: 22362), LC-SPDP (CatalogNo: 21651), MBS (Catalog No: 22311), SATA (Catalog No: 26102), SATP(Catalog No: 26100), SBAP (Catalog No: 22339), SIA (Catalog No: 22349),SIAB (Catalog No: 22329), SMCC (Catalog No: 22360), SMPB (Catalog No:22416), SMPH (Catalog No. 22363), SMPT (Catalog No: 21558), SPDP(Catalog No: 21857), Sulfo-EMCS (Catalog No: 22307), Sulfo-GMBS (CatalogNo: 22324), Sulfo-KMUS (Catalog No: 21111), Sulfo-LC-SPDP (Catalog No:21650), Sulfo-MBS (Catalog No: 22312), Sulfo-SIAB(Catalog No: 22327),Sulfo-SMCC (Catalog No: 22322), Sulfo-SMPB (Catalog No: 22317),Sulfo-LC-SMPT (Catalog No.: 21568).

Additionally, or alternatively, peptide or protein antigens can also beconjugated to flagellin components or Toll-like Receptor agonistcomponents of the invention via at least one lysine residue on bothconjugate partners. The two conjugate partners are combined along with ahomo-bifunctional amine-specific crosslinker. The appropriatehetero-conjugate is then purified away from unwanted aggregates andhomo-conjugates. Exemplary reagents for use in the conjugation methodscan be purchased from Pierce (Rockland, Ill.), for example, BSOCOES(Catalog No: 21600), BS₃ (Catalog No: 21580), DFDNB (Catalog No: 21525),DMA (Catalog No: 20663), DMP (Catalog No: 21666), DMS (Catalog No:20700), DSG (Catalog No: 20593), DSP (Catalog No: 22585), DSS (CatalogNo: 21555), DST (Catalog No: 20589), DTBP (Catalog No: 20665), DTSSP(Catalog No: 21578), EGS (Catalog No: 21565), MSA (Catalog No: 22605),Sulfo-DST (Catalog No: 20591), Sulfo-EGS (Catalog No: 21566), THPP(Catalog No: 22607).

Similarly, peptide or protein antigens can be conjugated to flagellincomponents or Toll-like Receptor agonist components of the invention viaat least one carboxyl group (e.g., glutamic acid, aspartic acid, or thecarboxy-terminus of the peptide or protein) on one partner and amines onthe other partner. The two conjugation partners are mixed together alongwith the appropriate heterobifunctional crosslinking reagent. Theappropriate hetero-conjugate is then purified away from unwantedaggregates and homo-conjugates. Exemplary reagents for use in theconjugation methods can be purchased from Pierce (Rockland, Ill.), forexample, AEDP (Catalog No: 22101), EDC (Catalog No: 22980) and TFCS(Catalog No: 22299).

In addition, carbohydrate antigens can be conjugated to proteins,flagellin components, flagellin, Toll-like Receptor agonist componentsand Toll-like Receptor agonists of the invention via at least onecysteine residue in the protein employing well-established techniques.For example, the protein is initially derivatized with aheterobifunctional crosslinker containing at least onesulfhydryl-specific group and at least one hydrazide group. Thepolysaccharide or oligosaccharide is then treated with an oxidizingagent such as sodium meta-periodate to generate terminal aldehydegroups. The oxidized carbohydrate is then added to the derivatizedprotein and conjugation to the aldehyde occurs via the hydrazide on thecrosslinker. Exemplary reagents for use in the conjugation methods canbe purchased from Pierce (Rockland, Ill.), for example, BMPH (CatalogNo: 22297), EMCH (Catalog No: 22106), KMUH (Catalog No: 22111) and PDPH(Catalog No: 22301).

Further lipopeptides can be conjugated to protein antigens, flagellincomponents or Toll-like Receptor components or Toll-like Receptoragonists of the invention via amino-acid side chains on the peptidechain. Similar strategies and reagents, as described above for peptideconjugation to proteins, would be employed.

TLRs can be activated by nucleic acids. For example, TLR3 is activatedby double-stranded (ds) RNA; TLR7 and TLR8 are activated bysingle-stranded (ss) RNA; and TLR9 is activated by CpG DNA sequences.Several different techniques can be employed to conjugate Toll-likeReceptor agonist components of nucleic acid TLRs to protein antigens.For example, for un-modified DNA molecule, the 5′ phosphate group can bemodified with the water-soluble carbodiimide EDC (Pierce; Rockford,Ill., Catalog No: 22980) followed by imidazole to form a terminalphosphoylimidazolide. A terminal amine can then be substituted for thisreactive group by the addition of ethylenediamine. The amine-modifiednucleic acid can then be conjugated to a cysteine on a protein using aheterobifunctional maleimide (cysteine-specific) and NHS-ester(lysine-specific) crosslinker, as described above for peptide-proteinconjugation.

Alternatively, or additionally, a sulfhydryl group can be incorporatedat the 5′ end by substituting cystamine for ethylenediamine in thesecond step. After reduction of the disulfide bond, the free sulfhydrylcan be conjugated to cysteines on proteins, flagellin components,flagellin, Toll-like Receptor agonist components and Toll-like Receptoragonists employing a homo-bifunctional maleimide-based crosslinker, orto lysines using a heterobifunctional crosslinker, as described above.Alternately, or additionally, synthetic oligonucleotides can besynthesized with modified bases on either the 5′ or 3′ end. Thesemodified bases can include primary amine or sulfhydryl groups which canbe conjugated to proteins using the methods described above. The 3′ endof RNA molecules may be chemically modified to allow coupling withproteins or other macromolecules. The diol on the 3′-ribose residue maybe oxidized using sodium meta-periodate to produce an aldehyde group.The aldehyde may then be conjugated to proteins using ahydrazide-containing crosslinker, such as MPBH (Pierce; Rockland, Ill.,Catalog No: 22305), which covalently modifies the carbonyl group, andthen conjugates to free thiols on proteins via a maleimide group.Alternatively, the 3′ hydroxyl group may be derivatized directly with anisocyanate-containing crosslinker, such as PMPI (Pierce; Rockland, Ill.,Catalog No: 28100), which also contains a maleimide group forconjugation to protein sulfhydryls.

Synthetic small-molecule TLR ligands (e.g., Toll-like Receptor agonists,Toll-like Receptor antagonists) have been identified. For example,imiquimod, which potently activates TLR7, and resiquimod, an activatorof both TLR7 and TLR8. Analogs of these compounds with varying levels ofpotency and specificity have been synthesized. The ability to conjugatea small molecule TLR agonist to an antigen of interest can depend on thechemical nature of the TLR ligand. Some TLR ligands may have activegroups that can be exploited for chemical conjugation. For example,imiquimod, as well as its analog gardiquimod (InvivoGen; San Diego,Calif.) and the TLR7-activating adenine analog CL087 (InvivoGen, SanDiego, Calif.), have primary amine groups (—NH₂), which can be targetsfor derivatization by crosslinkers containing imidoesters or NHS esters.In another strategy, gardiquimod contains an exposed hydroxyl (—OH)group, which can be derivatized by an isocyanate-containing crosslinker,such as PMPI (Pierce; Rockland, Ill. Catalog No: 28100) for subsequentcrosslinking to protein sulfhydryl groups.

In addition, custom synthesis of derivatives of small-molecule TLRligands can be arranged in order to attach novel functional groups atdifferent positions in the molecule to facilitate crosslinking Customderivatives can also include groups, such as maleimides, which can thenbe used for direct linking to protein antigens.

Chemical conjugation of an antigen to the flagellin component can resultin increased aqueous solubility of the antigen (e.g., an essentiallyhydrophobic antigen, such as a maturational cleavage site antigen) as acomponent of the composition.

The composition comprising a flagellin component that is at least aportion of a flagellin, wherein the flagellin component includes atleast one cysteine residue and whereby the flagellin component activatesa Toll-like Receptor 5 can include a cysteine residue in thehypervariable region of the flagellin component.

At least one cysteine residue substitutes for at least one amino acid ina naturally occurring flagellin amino acid sequence flagellin component.The cysteine residue can substitute for at least one amino acid selectedfrom the group consisting of amino acid 1, 237, 238, 239, 240, 241 and495 of SEQ ID NO: 812; at least one amino acid selected from the groupconsisting of amino acid 1, 240, 241, 242, 243, 244 and 505 of SEQ IDNO: 498; at least one amino acid selected from the group consisting ofamino acid 1, 237, 238, 239, 240, 241 and 504 of SEQ ID NO: 504; atleast one amino acid selected from the group consisting of amino acid 1,211, 212, 213 and 393 of SEQ ID NO: 815; at least one amino acidselected from the group consisting of amino acid 1, 151, 152, 153, 154and 287 of SEQ ID NO: 820; at least one amino acid selected from thegroup consisting of amino acid 1, 238, 239, 240, 241, 242, 243 and 497of SEQ ID NO: 502; at least one amino acid selected from the groupconsisting of amino acid 1, 237, 238, 239, 240, 241 and 495 of SEQ IDNO: 812.

The flagellin component or Toll-like Receptor agonist component caninclude at least a portion of a naturally occurring flagellin amino acidsequence in combination with the cysteine residue.

The composition of the invention wherein the cysteine residuesubstitutes for at least one amino acid in a naturally occurringflagellin amino acid sequence flagellin component, or wherein theflagellin component includes at least a portion of a naturally occurringflagellin amino acid sequence in combination with the cysteine residue,can activate a Toll-like Receptor 5. For example, a cysteine residue canbe placed within the D1/D2 domain proximate to the amino-terminus andcarboxy-terminus, remote to the TLR5 recognition site (see, for example,FIGS. 75 and 76). Alternatively, or additionally, the cysteine residuecan be placed at the distal point of the hypervariable domain (see, forexample, FIGS. 75 and 76) at about amino acid 237, about 238, about 239,about 240 and about 241 of SEQ ID NO: 812. Substituting polar or chargedamino acids is preferable to substituting hydrophobic amino acids withcysteine residues. Substitution within the TLR5 recognition site isleast preferable.

Flagellin from Salmonella typhimurium STF1 (FliC) is depicted in SEQ IDNO: 812 (Accession No: P06179). The TLR5 recognition site is amino acidabout 79 to about 117 and about 408 to about 439. Cysteine residues cansubstitute for or be included in combination with amino acid about 408to about 439 of SEQ ID NO: 812; amino acids about 1 and about 495 of SEQID NO: 812; amino acids about 237 to about 241 of SEQ ID NO: 812; and/oramino acids about 79 to about 117 and about 408 to about 439 of SEQ IDNO: 812.

Salmonella typhimurium flagellin STF2 (FljB) is depicted in SEQ ID NO:498. The TLR5 recognition site is amino acids about 80 to about 118 andabout 420 to about 451 of SEQ ID NO: 498. Cysteine residues cansubstitute for or be included in combination with amino acids about 1and about 505 of SEQ ID NO: 498; amino acids about 240 to about 244 ofSEQ ID NO: 498; amino acids about 79 to about 117 and/or about 419 toabout 450 of SEQ ID NO: 498.

Salmonella muenchen flagellin is depicted in SEQ ID NO: 504 (AccessionNo: #P06179). The TLR5 recognition site is amino acids about 79 to about117 and about 418 to about 449 of SEQ ID NO: 504. Cysteine residues cansubstitute for or be included in combination with amino acids about 1and about 504 of SEQ ID NO: 504; about 237 to about 241 of SEQ ID NO:504; about 79 to about 117; and/or about 418 to about 449 of SEQ ID NO:504.

Escherichia coli flagellin is depicted in SEQ ID NO: 502 (Accession No:P04949). The TLR5 recognition site is amino acids about 79 to about 117and about 410 to about 441 of SEQ ID NO: 502. Cysteine residues cansubstitute for or be included in combination with amino acids about 1and about 497 of SEQ ID NO: 502; about 238 to about 243 of SEQ ID NO:502; about 79 to about 117; and/or about 410 to about 441 of SEQ ID NO:502.

Pseudomonas auruginosa flagellin is depicted in SEQ ID NO: 815. The TLR5recognition site is amino acids about 79 to about 114 and about 308 toabout 338 of SEQ ID NO: 815. Cysteine residues can substitute for or beincluded in combination with amino acids about 1 and about 393 of SEQ IDNO: 815; about 211 to about 213 of SEQ ID NO: 815; about 79 to about114; and/or about 308 to about 338 of SEQ ID NO: 815.

Listeria monocytogenes flagellin is depicted in SEQ ID NO: 820. The TLR5recognition site is amino acids about 78 to about 116 and about 200 toabout 231 of SEQ ID NO: 820. Cysteine residues can substitute for or beincluded in combination with amino acids about 1 and about 287 of SEQ IDNO: 820; about 151 to about 154 of SEQ ID NO: 820; about 78 to about116; and/or about 200 to about 231 of SEQ ID NO: 820.

Experimentally defined TLR5 recognition sites on STF2 have beendescribed (see, for example, Smith, K. D., et al., Nature Immunology4:1247-1253 (2003) at amino acids about 79 to about 117 and about 420 toabout 451. In addition, Smith, K. D., et al., Nature Immunology4:1247-1253 (2003), based on sequence homology, identified TLR5recognition sites on other flagellins, such as STF1 at amino acids about79 to about 117, about 408 to about 439; P. aeruginosa at amino acidsabout 79 to about 117, about 308 to about 339; L. pneumophila at aminoacids about 79 to about 117, about 381 to about 419; E. coli at aminoacids about 79 to about 117, about 477, about 508; S. marcesens at aminoacids about 79 to about 117, about 265-about 296; B. subtilus at aminoacids about 77 to about 117, about 218 to about 249; and L.monocytogenes at amino acids about 77 to about 115, about 200 to about231.

The high-resolution structure STF1 (FliC) (SEQ ID NO: 812) has beendetermined and can be a basis for analysis of TLR5 recognition by aflagellin and the location of cysteine substitutions/additions.Flagellin resembles a “boomerang,” with the amino- and carboxy-terminiat the end of one arm (see, for example FIG. 77). The TLR5 recognitionsite is located roughly on the outer side of the boomerang just belowthe bend toward the amino- and carboxy-termini of the flagellin. Thehinge region, which is not required for TLR5 recognition, is locatedabove the bend. The region of greatest sequence homology of flagellinsis in the TLR5 recognition site. The next region of sequence homology isin the D1 and D2 domains, which include the TLR5 recognition site andthe amino- and carboxy-termini. The D1 and D2 domains, with or without alinker, is STF24Δ □(□□SEQ ID NO: 500), which can activate TLR5. Theregion of least sequence homology between flagellins is thehypervariable region.

It is believed that the ability of the flagellin component or Toll-likeReceptor agonist component to activate TLR5 can be accomplished bymaintaining the conjugation sites (cysteine residues substituted for atleast one amino acid in a naturally occurring flagellin amino acidsequence flagellin component or at least a portion of a naturallyoccurring flagellin amino acid sequence in combination with the cysteineresidue) remote from the TLR5 or TLR recognition site. For example, forSTF1 (SEQ ID NO: 812), for which a high resolution structuraldetermination is available, this may be achieved in the D1 domain, D2domain or in the hinge region. In the D1/D2 domain the amino- andcarboxy-termini can be remote (also referred to herein as “distal”) fromthe TLR5 recognition site, and moving away from either the amino orcarboxy terminus may bring the conjugation site closer to therecognition site and may interfere with TLR5 activity. In the hingeregion amino acids about 237 to about 241 of SEQ ID NO: 812, areapproximately at the other tip of the “boomerang” and are about the samedistance from the TLR5 recognition site as the amino- andcarboxy-termini. This site may also be a location that maintains TLR5recognition.

Amino acid identity can be taken into consideration for the location ofconjugation sites. Polar and charged amino acids (e.g., serine, asparticacid, lysine) are more likely to be surface exposed and amenable toattachment of an antigen. Hydrophobic amino acids (e.g., valine,phenalalanine) are more likely to be buried and participate instructural interactions and should be avoided.

Compositions that include flagellin components with cysteine residues orToll-like Receptor agonist components with cysteine residues activateTLR5 and can be chemically conjugated to antigens.

The compositions and methods of employing the compositions of theinvention can further include a carrier protein. The carrier protein canbe at least one member selected from the group consisting of a tetanustoxoid, a Vibrio cholerae toxoid, a diphtheria toxoid, a cross-reactivemutant of diphtheria toxoid, a E. coli B subunit of a heat labileenterotoxin, a tobacco mosaic virus coat protein, a rabies virusenvelope protein, a rabies virus envelope glycoprotein, a thyroglobulin,a heat shock protein 60, a keyhole limpet hemocyanin and an earlysecreted antigen tuberculosis-6.

The composition comprising a flagellin component that is at least aportion of a flagellin, wherein the flagellin component includes atleast one cysteine residue and whereby the flagellin component activatesa Toll-like Receptor 5 can include at least one lysine of the flagellincomponent that has been substituted with at least one member selectedfrom the group consisting of an arginine residue, a serine residue and ahistidine residue.

In an additional embodiment, the invention is a composition comprising aToll-like Receptor agonist component that is at least a portion of aToll-like Receptor agonist, wherein the Toll-like Receptor agonistcomponent includes at least one cysteine residue in a position where acysteine residue does not occur in the native Toll-like Receptoragonist, whereby the Toll-like Receptor agonist component activates aToll-like Receptor. “Component,” as used herein in reference to aToll-like Receptor agonist component, means at least part of or theentirety of a Toll-like Receptor agonist.

In one embodiment, the cysteine residue in the composition comprising aToll-like Receptor agonist component that is at least a portion of aToll-like Receptor agonist, wherein the Toll-like Receptor agonistcomponent includes at least one cysteine residue in a position where acysteine residue does not occur in the native Toll-like Receptoragonist, whereby the Toll-like Receptor agonist component activates aToll-like Receptor, substitutes for at least one amino acid in anaturally occurring amino acid sequence of a Toll-like Receptor agonistcomponent. The cysteine can substitute for at least one amino acidremote to the Toll-like Receptor recognition site of the Toll-likeReceptor agonist component.

In another embodiment, the Toll-like Receptor agonist component includesat least a portion of a naturally occurring Toll-like Receptor agonistamino acid sequence in combination with a cysteine residue. The cysteineresidue in combination with the naturally occurring Toll-like Receptoragonist can be remote to the Toll-like Receptor recognition site of theToll-like Receptor agonist component.

The composition comprising a Toll-like Receptor agonist component thatis at least a portion of a Toll-like Receptor agonist, wherein theToll-like Receptor agonist component includes at least one cysteineresidue in a position where a cysteine residue does not occur in thenative Toll-like Receptor agonist, whereby the Toll-like Receptoragonist component activates a Toll-like Receptor can further include atleast a portion of at least one antigen (e.g., an influenza antigen,such as a influenza integral membrane protein antigen, HA, HA1-1, HA1-2,M2, M2e).

In still another embodiment, the invention is a composition comprising aflagellin component that is at least a portion of a flagellin, whereinat least one lysine of the flagellin component has been substituted withat least one arginine, whereby the flagellin component activates aToll-like Receptor 5.

“Substituted,” as used herein in reference to the flagellin, flagellincomponent, Toll-like Receptor agonist or Toll-like Receptor agonistcomponent, means that at least one amino acid, such as a lysine of theflagellin component, has been modified to another amino acid residue,for example, a conservative substitution (e.g., arginine, serine,histidine) to thereby form a substituted flagellin component orsubstituted Toll-like Receptor agonist component. The substitutedflagellin component or substituted Toll-like Receptor agonist componentcan be made by generating recombinant constructs that encode flagellinwith the substitutions, by chemical means, by the generation of proteinsor peptides of at least a portion of the flagellin by protein synthesistechniques, or any combination thereof.

The lysine residue that is substituted with an amino acid (e.g.,arginine, serine, histidine) can be at least one lysine residue selectedfrom the group consisting of lysine 19, 41, 58, 135, 160, 177, 179, 203,215, 221, 228, 232, 241, 251, 279, 292, 308, 317, 326, 338, 348, 357,362, 369, 378, 384, 391 and 410 of SEQ ID NO: 812.

The flagellin can be a S. typhimurium flagellin that includes SEQ ID NO:816. The lysine residue that is substituted with an amino acid (e.g.,arginine, serine, histidine) can be at least one lysine residue selectedfrom the group consisting of lysine 20, 42, 59, 136, 161, 177, 182, 189,209, 227, 234, 249, 271, 281, 288, 299, 319, 325, 328, 337, 341, 355,357, 369, 381, 390, 396, 403, 414 and 422 of SEQ ID NO: 816.

The flagellin can be an E. coli fliC that includes SEQ ID NO: 814. Theflagellin can be a S. muenchen that include the includes SEQ ID NO: 813.The flagellin can be a P. aeruginosa flagellin that includes SEQ ID NO:815. The flagellin can be a Listeria monocytogenes flagellin thatincludes SEQ ID NO: 820.

The compositions of the invention can include a flagellin that haslysines substituted in a region adjacent to the motif C of theflagellin, the motif N of the flagellin, both the motif C and the motifN of the flagellin, domain 1 of the flagellin, domain 2 of the flagellinor any combination thereof. Motif C and motif N of flagellin and domain1 and domain 2 of the flagellin can be involved in activation of TLR5 bythe flagellin (Murthy, et al., J. Biol. Chem., 279:5667-5675 (2004)).

Chemical conjugation of a protein, peptide, or polypeptide to anothermolecule can be by derivatization of a secondary group, such as alysine. Certain lysine residues in flagellin are near or in domain 1,the motif C or motif N, motifs can be important in binding of theflagellin to TLR5. For example, lysine residues at amino acids 58, 135,160 and 410 of SEQ ID NO: 812 may be substituted with at least onemember selected from the group consisting of an arginine residue, aserine residue and a histidine residue. Derivatization of such lysineresidues to, for example, chemically conjugated antigens to flagellins,may decrease the ability or the binding affinity of the flagellin toTLR5 and, thus, diminish an innate immune response mediated by TLR5.Substitution of at least one lysine residue in a flagellin that may benear to regions of the flagellin that are important in mediatinginteractions with TLR5 (e.g., motif C, motif N, domain 1) with anotheramino acid (e.g., arginine, serine, histidine) may preserve or enhanceflagellin binding to TLR5. In a particular embodiment, the amino acidsubstitution is a conservative amino acid substitution with at least onemember selected from the group consisting of arginine, serine andhistidine. Exemplary commercially available reagents for chemicalconjugation are described herein.

Certain lysine residues in flagellin are in the domain (domain 1) andcan be important for activation of TLR5. For example, lysine residues atpositions 58, 135, 160 and 410 of SEQ ID NO: 812 are in domain 1.Derivatization of such lysine residues to, for example, chemicallyconjugated antigens, may decrease TLR5 bioactivity and, thus, diminishan innate immune response mediated by TLR5.

Lysine residues that can be substituted can include lysine residuesimplicated in TLR5 activation. Lysine residues in motif N (amino acids95-108 of SEQ ID NO: 812) and/or motif C (amino acids 441-449 of SEQ IDNO: 812) can be suitable for substitution. Substitution of certainlysine residue in the flagellin (e.g., lysine at amino acid position 19,41) with, for example, an arginine, serine or histidine, can maintainbinding of the flagellin to TLR5 and leave other lysines available forchemical conjugate to another molecule, such as an antigen (e.g.,protein) or another molecule, such as another protein, peptide orpolypeptide.

The X-ray crystal structure of the F41 fragment of flagellin fromSalmonella typhimurium shows the domain structure of flagellin (Samatey,F. A., et al., Nature 410:321 (2001)). The full length flagellin proteincontains 4 domains, designated as D0, D1, D2 and D3. Three of thesedomains are shown in the crystal structure because the structure wasmade with a proteolytic fragment of full length flagellin. The aminoacid sequences of Salmonella typhimurium flagellin for these regions,numbered relative to SEQ ID NO: 812 are as follows:

D0 contains the regions A1 through A55 and S451 through R494

D1 contains the regions N56 through Q176 and T402 through R450

D2 contains the regions K177 through G189 and A284 through A401

D3 contains the region Y190 though V283

Exemplary lysine residues of SEQ ID NO: 812 suitable for substitutionwith, for example, arginine, histidine, or serine, can include:

D0 contains 2 lysine residues; K19, K41

D1 contains 4 lysine residues; K58, K135, K160 and K410

D2 contains 14 lysine residues at positions 177, 179, 292, 308, 317,326, 338, 348, 357, 362, 369, 378, 384, 391

D3 contains 8 lysine residues at positions 203, 215, 221, 228, 232, 241,251, 279

Exemplary lysine residues suitable for substitution include lysines atpositions 58, 135, 160 and 410 of SEQ ID NO: 812 (Jacchieri, S. G., et.al., J. Bacteriol. 185:4243 (2003); Donnelly, M. A., et al., J. Biol.Chem. 277:40456 (2002)). The sequences were obtained from the Swiss-ProtProtein Knowledgebase located online at http://us.expasy.org/sprot/.Lysine residues that can be modified are indicated with a *.

Exemplary lysine residues of SEQ ID NO: 816 suitable for substitutioncan include:

D0—with two lysines at positions 20, 42;

D1—with five lysines at positions 59, 136, 161, 414, 422;

D2—with sixteen lysines at positions 177, 182, 189, 299, 319, 325, 328,337, 341, 355, 357, 369, 381, 390, 396, 403 and

D3—with seven lysines at positions 209, 227, 234, 249, 271, 281, 288.

In a further embodiment, the invention is a composition comprising aflagellin component that is at least a portion of a flagellin, whereinat least one lysine of the flagellin component has been substituted withat least one histidine residue, whereby the flagellin componentactivates a Toll-like Receptor 5.

In yet another embodiment, the invention is a method of stimulating animmune response in a subject, comprising the step of administering tothe subject a composition that includes a flagellin component that is atleast a portion of a flagellin, wherein the flagellin component includesat least one cysteine residue and whereby the flagellin componentactivates a Toll-like Receptor 5.

In another embodiment, the invention is a method of stimulatingprotective immunity in a subject, comprising the step of administeringto the subject a composition that includes a flagellin component that isat least a portion of a flagellin, wherein the flagellin componentincludes at least one cysteine residue and whereby the flagellincomponent activates a Toll-like Receptor 5.

In yet another embodiment, the invention is a method of stimulatingprotective immunity in a subject, comprising the step of administeringto the subject a composition that includes a flagellin component that isat least a portion of a flagellin, wherein at least one lysine of theflagellin component has been substituted with at least one arginine,whereby the flagellin component activates a Toll-like Receptor 5.

In still another embodiment, the invention is a method of stimulating animmune response in a subject, comprising the step of administering tothe subject a composition that includes a flagellin component that is atleast a portion of a flagellin, wherein at least one lysine of theflagellin component has been substituted with at least one serineresidue, whereby the flagellin component activates a Toll-like Receptor5.

In another embodiment, the invention is a method of stimulating aprotective immune response in a subject, comprising the step ofadministering to the subject a composition that includes a flagellincomponent that is at least a portion of a flagellin, wherein at leastone lysine of the flagellin component has been substituted with at leastone serine residue, whereby the flagellin component activates aToll-like Receptor 5.

In an additional embodiment, the invention is a method of stimulating animmune response in a subject, comprising the step of administering tothe subject a composition that includes a flagellin component that is atleast a portion of a flagellin, wherein at least one lysine of theflagellin component has been substituted with at least one histidineresidue, whereby the flagellin component activates a Toll-like Receptor5.

In a further embodiment, the invention is a method of stimulatingprotective immunity in a subject, comprising the step of administeringto the subject a composition that includes a flagellin component that isat least a portion of a flagellin, wherein at least one lysine of theflagellin component has been substituted with at least one histidineresidue, whereby the flagellin component activates a Toll-like Receptor5.

Another embodiment of the invention is a method of stimulating an immuneresponse in a subject, comprising the step of administering to thesubject a composition that includes a Toll-like Receptor agonistcomponent that is at least a portion of a Toll-like Receptor agonist,wherein the Toll-like Receptor agonist component includes at least onecysteine residue in a position where a cysteine residue does not occurin the native Toll-like Receptor agonist, whereby the Toll-like Receptoragonist component activates a Toll-like Receptor agonist.

Another embodiment of the invention is a method of stimulating aprotective immunity in a subject, comprising the step of administeringto the subject a composition that includes a Toll-like Receptor agonistcomponent that is at least a portion of a Toll-like Receptor agonist,wherein the Toll-like Receptor agonist component includes at least onecysteine residue in a position where a cysteine residue does not occurin the native Toll-like Receptor agonist, whereby the Toll-like Receptoragonist component activates a Toll-like Receptor agonist.

In yet another embodiment, the invention is a composition comprising anantigen component that includes at least a portion of a hemagglutininmaturational cleavage site and an agonist component that includes aToll-like Receptor agonist (e.g., at least one member selected from thegroup consisting of a Toll-like Receptor 1 agonist, a Toll-like Receptor3 agonist, a Toll-like Receptor 4 agonist, a Toll-like Receptor 6agonist, a Toll-like Receptor 7 agonist and a Toll-like Receptor 9agonist) wherein the Toll-like Receptor agonist is not a Toll-likeReceptor 2 agonist (e.g., the outer membrane protein complex (OMPC),such as OMPC of Neiserria meningitides). The hemagglutinin maturationalcleavage site can include at least one member selected from the groupconsisting of an influenza A hemagglutinin maturational cleavage site,an influenza B hemagglutinin maturational cleavage site and an influenzaC hemagglutinin maturational cleavage site.

In one embodiment, the antigen component and the agonist component ofthe compositions of the invention can be components of a fusion protein.In another embodiment, the antigen component is chemically conjugated tothe agonist component.

The agonist component includes a composition comprising a flagellincomponent that is at least a portion of a flagellin, wherein theflagellin component includes at least one cysteine residue and wherebythe flagellin component activates a Toll-like Receptor 5. The antigencomponent further includes at least a portion of a matrix 2 protein,which can further include a second agonist component that includes atleast a portion of a second Toll-like Receptor agonist (e.g., at least aportion of at least one member selected from the group consisting of aToll-like Receptor 2 agonist, a Toll-like Receptor 3 agonist, aToll-like Receptor 4 agonist, a Toll-like Receptor 5 agonist, aToll-like Receptor 6 agonist, a Toll-like Receptor 7 agonist and aToll-like Receptor 9 agonist). The matrix-2 protein can be fused to thesecond agonist component or chemically conjugated to a second agonistcomponent.

An “antigen component,” as used herein, refers to a part or the entiretyof an antigen.

An “agonist component” as used herein, refers to a part or the entiretyof an agonist, such as a Toll-like Receptor agonist.

In an additional embodiment, the invention is a method of stimulating animmune response in a subject, comprising the step of administering tothe subject a composition comprising an antigen component that includesat least a portion of a hemagglutinin maturational cleavage site and anagonist component that includes a Toll-like Receptor agonist, whereinthe Toll-like Receptor agonist is not a Toll-like Receptor 2 agonist.

In a further embodiment, the invention is a method of stimulatingprotective immunity in a subject, comprising the step of administeringto the subject a composition comprising an antigen component thatincludes at least a portion of a hemagglutinin maturational cleavagesite and an agonist component that includes a Toll-like Receptoragonist, wherein the Toll-like Receptor agonist is not a Toll-likeReceptor 2 agonist.

In yet another embodiment, the invention is a composition that includesat least a portion of a HA antigen (e.g., the maturational cleavagesite) that is fused (e.g., recombinantly) to or chemically conjugated toat least of portion of a hinge region of a flagellin, a flagellincomponent or in a flagellin in which the hinge region has been deleted;and at least a portion of an M2e protein that is fused (e.g.,recombinantly) to or chemically conjugated to a flagellin or a flagellincomponent, for example, at the amino- or carboxy-terminus of theflagellin or the flagellin component (See, for example, FIG. 77).

In an additional embodiment, the invention includes a protein, peptidepolypeptide having at least about 70%, at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,at least about 98% and at least about 99% sequence identity to theproteins, polypeptides and peptides of the invention.

The percent identity of two amino acid sequences (or two nucleic acidsequences) can be determined by aligning the sequences for optimalcomparison purposes (e.g., gaps can be introduced in the sequence of afirst sequence). The amino acid sequence or nucleic acid sequences atcorresponding positions are then compared, and the percent identitybetween the two sequences is a function of the number of identicalpositions shared by the sequences (i.e., % identity=# of identicalpositions/total # of positions×100). The length of the protein ornucleic acid encoding can be aligned for comparison purposes is at least30%, preferably, at least 40%, more preferably, at least 60%, and evenmore preferably, at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or100%, of the length of the reference sequence, for example, the nucleicacid sequence of a protein portion of HA (e.g., HA 1-1, HA 1-2), fusionprotein, antigen, or polypeptide.

The actual comparison of the two sequences can be accomplished bywell-known methods, for example, using a mathematical algorithm. Apreferred, non-limiting example of such a mathematical algorithm isdescribed in Karlin et al. (Proc. Natl. Acad. Sci. USA, 90:5873-5877(1993), the teachings of which are hereby incorporated by reference inits entirety). Such an algorithm is incorporated into the BLASTN andBLASTX programs (version 2.2) as described in Schaffer et al. (NucleicAcids Res., 29:2994-3005 (2001), the teachings of which are herebyincorporated by reference in its entirety). When utilizing BLAST andGapped BLAST programs, the default parameters of the respective programs(e.g., BLASTN; available at the Internet site for the National Centerfor Biotechnology Information) can be used. In one embodiment, thedatabase searched is a non-redundant (NR) database, and parameters forsequence comparison can be set at: no filters; Expect value of 10; WordSize of 3; the Matrix is BLOSUM62; and Gap Costs have an Existence of 11and an Extension of 1.

Another mathematical algorithm employed for the comparison of sequencesis the algorithm of Myers and Miller, CABIOS (1989), the teachings ofwhich are hereby incorporated by reference in its entirety. Such analgorithm is incorporated into the ALIGN program (version 2.0), which ispart of the GCG (Accelrys, San Diego, Calif.) sequence alignmentsoftware package. When utilizing the ALIGN program for comparing aminoacid sequences, a PAM120 weight residue table, a gap length penalty of12, and a gap penalty of 4 is used. Additional algorithms for sequenceanalysis are known in the art and include ADVANCE and ADAM as describedin Torellis and Robotti (Comput. Appl. Biosci., 10: 3-5 (1994), theteachings of which are hereby incorporated by reference in itsentirety); and FASTA described in Pearson and Lipman (Proc. Natl. Acad.Sci. USA, 85: 2444-2448 (1988), the teachings of which are herebyincorporated by reference in its entirety).

The percent identity between two amino acid sequences can also beaccomplished using the GAP program in the GCG software package(Accelrys, San Diego, Calif.) using either a Blossom 63 matrix or aPAM250 matrix, and a gap weight of 12, 10, 8, 6, or 4 and a lengthweight of 2, 3, or 4. In yet another embodiment, the percent identitybetween two nucleic acid sequences can be accomplished using the GAPprogram in the GCG software package (Accelrys, San Diego, Calif.), usinga gap weight of 50 and a length weight of 3.

The nucleic acid sequence encoding a protein portion of HA, polypeptideor fusion proteins of the invention and polypeptides of the inventioncan include nucleic acid sequences that hybridize to nucleic acidsequences or complements of nucleic acid sequences of the invention, forexample, SEQ ID NOS: 53-58, 64, 68, 71, 72, 73, 76, 78, 80, 84, 87, 104,107, 110, 111, 112, 115, 116, 117, 119, 120, 121, 122, 123, 124, 125,126, 127, 128, 129, 130, 131, 132, 133, 137, 139, 142, 143, 146, 147,150, 167, 184, 185, 186, 187, 188, 189, 190, 191, and 192); and nucleicacid sequences that encode amino acid sequences and fusion proteins ofthe invention (e.g., SEQ ID NOS: 89-92, 95, 151-160, 177, 209, 210 and211) under selective hybridization conditions (e.g., highly stringenthybridization conditions). As used herein, the terms “hybridizes underlow stringency,” “hybridizes under medium stringency,” “hybridizes underhigh stringency,” or “hybridizes under very high stringency conditions,”describe conditions for hybridization and washing of the nucleic acidsequences. Guidance for performing hybridization reactions, which caninclude aqueous and nonaqueous methods, can be found in Aubusel, F. M.,et al., Current Protocols in Molecular Biology, John Wiley & Sons, N.Y.(2001), the teachings of which are hereby incorporated herein in itsentirety.

For applications that require high selectivity, relatively highstringency conditions to form hybrids can be employed. In solutions usedfor some membrane based hybridizations, addition of an organic solvent,such as formamide, allows the reaction to occur at a lower temperature.High stringency conditions are, for example, relatively low salt and/orhigh temperature conditions. High stringency are provided by about 0.02M to about 0.10 M NaCl at temperatures of about 50° C. to about 70° C.High stringency conditions allow for limited numbers of mismatchesbetween the two sequences. In order to achieve less stringentconditions, the salt concentration may be increased and/or thetemperature may be decreased. Medium stringency conditions are achievedat a salt concentration of about 0.1 to 0.25 M NaCl and a temperature ofabout 37° C. to about 55° C., while low stringency conditions areachieved at a salt concentration of about 0.15 M to about 0.9 M NaCl,and a temperature ranging from about 20° C. to about 55° C. Selection ofcomponents and conditions for hybridization are well known to thoseskilled in the art and are reviewed in Ausubel et al. (1997, ShortProtocols in Molecular Biology, John Wiley & Sons, New York N.Y., Units2.8-2.11, 3.18-3.19 and 4-64.9).

A “subject,” as used herein, can be a mammal, such as a primate orrodent (e.g., rat, mouse). In a particular embodiment, the subject is ahuman.

An “effective amount,” when referring to the amount of a composition andfusion protein of the invention, refers to that amount or dose of thecomposition and fusion protein, that, when administered to the subjectis an amount sufficient for therapeutic efficacy (e.g., an amountsufficient to stimulate an immune response in the subject). Thecompositions and fusion proteins of the invention can be administered ina single dose or in multiple doses.

The methods of the present invention can be accomplished by theadministration of the compositions and fusion proteins of the inventionby enteral or parenteral means. Specifically, the route ofadministration is by oral ingestion (e.g., drink, tablet, capsule form)or intramuscular injection of the composition and fusion protein. Otherroutes of administration as also encompassed by the present inventionincluding intravenous, intradermal, intraarterial, intraperitoneal, orsubcutaneous routes, and nasal administration. Suppositories ortransdermal patches can also be employed.

The compositions and fusion proteins of the invention can beadministered ex vivo to a subject's autologous dendritic cells.Following exposure of the dendritic cells to the composition and fusionprotein of the invention, the dendritic cells can be administered to thesubject.

The compositions and fusion proteins of the invention can beadministered alone or can be coadministered to the patient.Coadministration is meant to include simultaneous or sequentialadministration of the composition, fusion protein or polypeptide of theinvention individually or in combination. Where the composition andfusion protein are administered individually, the mode of administrationcan be conducted sufficiently close in time to each other (for example,administration of the composition close in time to administration of thefusion protein) so that the effects on stimulating an immune response ina subject are maximal. It is also envisioned that multiple routes ofadministration (e.g., intramuscular, oral, transdermal) can be used toadminister the compositions and fusion proteins of the invention.

The compositions and fusion proteins of the invention can beadministered alone or as admixtures with conventional excipients, forexample, pharmaceutically, or physiologically, acceptable organic, orinorganic carrier substances suitable for enteral or parenteralapplication which do not deleteriously react with the extract. Suitablepharmaceutically acceptable carriers include water, salt solutions (suchas Ringer's solution), alcohols, oils, gelatins and carbohydrates suchas lactose, amylose or starch, fatty acid esters, hydroxymethycellulose,and polyvinyl pyrrolidine. Such preparations can be sterilized and, ifdesired, mixed with auxillary agents such as lubricants, preservatives,stabilizers, wetting agents, emulsifiers, salts for influencing osmoticpressure, buffers, coloring, and/or aromatic substances and the likewhich do not deleteriously react with the compositions, fusion proteinsor polypeptides of the invention. The preparations can also be combined,when desired, with other active substances to reduce metabolicdegradation. The compositions and fusion proteins of the invention canbe administered by is oral administration, such as a drink,intramuscular or intraperitoneal injection or intranasal delivery. Thecompositions and fusion proteins alone, or when combined with anadmixture, can be administered in a single or in more than one dose overa period of time to confer the desired effect (e.g., alleviate preventviral infection, to alleviate symptoms of virus infection, such asinfluenza or flaviviral infection).

When parenteral application is needed or desired, particularly suitableadmixtures for the compositions and fusion proteins are injectable,sterile solutions, preferably oily or aqueous solutions, as well assuspensions, emulsions, or implants, including suppositories. Inparticular, carriers for parenteral administration include aqueoussolutions of dextrose, saline, pure water, ethanol, glycerol, propyleneglycol, peanut oil, sesame oil, polyoxyethylene-block polymers, and thelike. Ampules are convenient unit dosages. The compositions, fusionproteins or polypeptides can also be incorporated into liposomes oradministered via transdermal pumps or patches. Pharmaceutical admixturessuitable for use in the present invention are well-known to those ofskill in the art and are described, for example, in PharmaceuticalSciences (17th Ed., Mack Pub. Co., Easton, Pa.) and WO 96/05309 theteachings of which are hereby incorporated by reference.

The compositions and fusion proteins of the invention can beadministered to a subject on a support that presents the compositions,fusion proteins and polypeptides of the invention to the immune systemof the subject to generate an immune response in the subject. Thepresentation of the compositions, fusion proteins and polypeptides ofthe invention would preferably include exposure of antigenic portions ofthe viral protein to generate antibodies. The components (e.g., PAMP anda viral protein) of the compositions, fusion proteins and polypeptidesof the invention are in close physical proximity to one another on thesupport. The compositions and fusion proteins of the invention can beattached to the support by covalent or noncovalent attachment.Preferably, the support is biocompatible. “Biocompatible,” as usedherein, means that the support does not generate an immune response inthe subject (e.g., the production of antibodies). The support can be abiodegradable substrate carrier, such as a polymer bead or a liposome.The support can further include alum or other suitable adjuvants. Thesupport can be a virus (e.g., adenovirus, poxvirus, alphavirus),bacteria (e.g., Salmonella) or a nucleic acid (e.g., plasmid DNA).

The dosage and frequency (single or multiple doses) administered to asubject can vary depending upon a variety of factors, including priorexposure to an antigen, a viral protein, the duration of viralinfection, prior treatment of the viral infection, the route ofadministration of the composition, fusion protein or polypeptide; size,age, sex, health, body weight, body mass index, and diet of the subject;nature and extent of symptoms of viral exposure, viral infection and theparticular viral responsible for the infection (e.g., a flavivirus,influenza virus), or treatment or infection of an other antigen, such asan influenza antigen, kind of concurrent treatment, complications fromthe viral exposure, viral infection or exposure or other health-relatedproblems. Other therapeutic regimens or agents can be used inconjunction with the methods and compositions, fusion proteins orpolypeptides of the present invention. For example, the administrationof the compositions and fusion proteins can be accompanied by otherviral therapeutics or use of agents to treat the symptoms of a conditionassociated with or consequent to exposure to the antigen, such asflavivirus infection (e.g., high fever, numbness, DHF,meningoencephalitis) or influenza infection, for example. Adjustment andmanipulation of established dosages (e.g., frequency and duration) arewell within the ability of those skilled in the art.

Influenza viruses are single-stranded RNA viruses that belong to theviral family Orthomyxoviridae. Influenza viruses are divided into threetypes (A, B, C) determined by the antigenic differences inribonucleoprotein (RNP) and matrix (M) antigens of the viruses.Influenza A virus naturally infects humans and several other mammalianspecies, including swine and horses, and a wide variety of avianspecies, and causes epidemics and pandemics in the human population.Influenza B virus appears to naturally infect only humans and can causeepidemics in humans. Influenza C virus has been isolated from humans andswine, but generally does not occur in epidemics and usually results inmild disease in humans.

Mature influenza virions are enveloped with a pleomorphic structureranging in diameter from 80 to 120 nm. The single-stranded RNA genome isclosely associated with a helical nucleoprotein and is present in seven(influenza C) or eight (influenza A and B) separate segments ofribonucleoprotein (RNP), each of which has to be present for successfulreplication of the virus. The segmented genome is enclosed within anouter lipoprotein envelope. Matrix protein 1 (MP1 or also referred toherein as “M1”) lines the inside of the outer lipoprotein envelope andis bound to the RNP.

Hemagglutinin (HA) is a surface glycoprotein on a virus (e.g., aninfluenza virus) that is responsible for binding to N-AcetylNeuraminicAcid (NeuNAc; also referred to herein as “sialic acid”) on host cellsand subsequent fusion of viral and host membranes. HA acquired its nameby virtue of its ability to cause red blood cells to clump, oragglutinate. Influenza HA is a trimer consisting of the three monomeric(HA0) subunits. HA performs two critical functions during the infectionprocess: binding to a cell surface sialyloligosaccharide receptor andfusion of virus and host cell membrane. Following binding of the HAtrimer to the plasma membrane of a host cell, the host cell membraneengulfs the virus in an endosome and attempts to digest the contents ofthe endosome by acidifying its interior and transferring it to alysosome in the host cell. However, the acidic environment of thelysosome destabilizes HA, resulting in partial unfolding of HA0 whichexposes a protease-sensitive site (the maturaional cleaveage site) thatis cleaved by a host protease to form HA1 and HA2 subunits which areconnected by a single disulfide bond (Wiley, D. C., et al., Annu. Rev.Biochem. 56:365-394 (1987)). Cleavage occurs at a specific amino acidresidue and generates a hydrophobic amino terminus for the HA2 subunit.This hydrophobic terminus of HA2 mediates fusion between the viralenvelope and the endosomal membrane of the host cell and releases thecontents of the virion into the cytoplasm of the cell, a process knownas uncoating. Thus, cleavage of the HA polypeptide is a requirement forinfectivity.

The crystal structure of several viral hemagglutinins has beendetermined (see, for example, Wilson, I. A., et al., Nature 289:366-373(1981); Chen, J., et al., Cell 95:409-417 (1998); Ha, Y., et al., TheEMBO Journal 21: 865-875 (2002); Russell, R. J., et al., Virology325:287-296 (2004); and Cox, N. J., et al., In: Toply and Wilson'sMicrobiology and Microbial Infections, eds. B. W. J. Mathy, et al., Vol.1 (9^(th) ed.) New York, N.Y., Oxford Univ. Press, Ch. 32, p. 634(1998)). X-ray crystallographic structures show that HA is folded intotwo structural components or domains—a globular head and a fibrous stalk(see, for example, FIG. 1). The globular head includes HA1, includingthat part of HA1 that binds to sialic acid (also referred to as the“receptor binding site or domain” or “sialic acid binding site ordomain”), and antiparallel β-sheets. The fibrous stalk is more proximalto the viral membrane and consists of all of HA2 and part of HA1,including the cleavage site between HA1 and HA2.

There are fifteen known subtypes of Influenza A HA (H1-H15) that sharebetween about 40 to about 60% sequence identity (World HealthOrganization BULL. World Health Organ., 58:585-591 (1980)). Influenzaviruses containing all 15 HA subtypes have been isolated from avianspecies (H5, H7, and H9), equine (H3 and H7), seals (H3, H4 and H7),whales (H1 and H13) and swine (H1, H3, and H9). Subtypes of influenza Avirus are generally named according to the particular antigenicdeterminants of HA (H, 15 major types) and neuraminidase (N, about 9major types). For example, subtypes include influenza A (H2N1), A(H3N2),A(H5N1), A(H7N2), A(H9N2), A(H1/H0), A(H3/H0) and A(H5/H0). In the lastcentury, three subtypes of influenza A resulted in pandemics: H1 in 1918and 1977; H2 in 1957 and H3 in 1968. In 1997, an H5 avian virus and in1999, an H9 virus resulted in outbreaks of respiratory disease in HongKong. HA from influenza type B viruses have been isolated from humansand seals and are not divided into subtypes.

A host infected with influenza can mount an antibody response to theglobular head of HA that protects that host from subsequent infectionwith the same strain of virus by blocking the interaction between HA andthe host cell, i.e., neutralizing the infectivity of the virus. Due tothe low fidelity and high rate of influenza RNA replication, the virusis constantly experiencing minor mutations in the HA gene that preservethe globular head structure and host cell interaction, but may allowprogeny virus to escape immune surveillance. These point mutations arereferred to as “antigenic drift.” In addition, if a single host issimultaneously infected with two different strains of influenza A, a newsubtype of virus may emerge as a result of reassortment, or the exchangeof the RNA segments, or genes, between different strains of influenza Aviruses. The viruses emerging from reassortment present the human immunesystem with a new antigenic experience that usually results in highmorbidity and mortality. This type of drastic antigenic change is knownas “antigenic shift.” Since type B influenza viruses circulate almostexclusively in humans, these viruses cannot undergo reassortment withanimal strains and, thus, are changed only by antigenic drift.

Immunity to HA can reduce the likelihood of infection and severity ofdisease if infection does occur. HA is an important antigenic target andthe efficacy of vaccines depends on the antigenic match between thevaccine strain and the circulating strain. Since the hemagglutininprotein readily undergoes antigenic shift and drift in order to evadethe host's immune defense, traditional vaccines must be based oncurrently circulating influenza strains and annually updated. Annualupdates of influenza vaccines are not only costly they also requiresignificant amounts of production time and manufacturing infrastructure.A vaccine composition based on invariant regions of the virus mayprovide broadly cross-reactive protection.

In contrast to the globular head of HA, changes in amino acid residuessurrounding the maturational cleavage site of HA are limited due tofunctional constraints. Amino acid residues surrounding the HAmaturational cleavage site influence recognition and thereforecleavability of the site by the host protease. Since the virus does notcode for the protease, changes in the amino acid residues surroundingthe maturational cleavage site are restricted. As a consequence apeptide of about 20 amino acids spanning the maturational cleavage siteremains genetically stable across influenza viruses of the same HAsubtype (WO 2004/080403; Bianchi, et al. J Virol 79:7380-7388 (2005)) oras branched peptides (Horvath, et al Immunol Letters 60:127-136 (1998),Nagy, et at Scand J Immunol 40:281-291 (1994)).

A second highly conserved antigen of influenza A is the ectodomain ofthe matrix 2 protein (M2e). M2 is a 97-amino acid protein expressed atlow levels in mature virions and much higher levels on infected cells.The M2 protein forms a homotetramer that functions as an ion channelwhich is critical to the replication of the virus, thus, mutations inM2e are not as well tolerated as mutations in HA. The 24-amino acidectodomain (M2e) is highly conserved across multiple influenza Astrains. In mammals, M2e is poorly immunogenic in its native form.Antibodies to M2e can confer passive protection in animal models ofinfluenza A infection (Treanor, J. J., et al., J Virol 64:1375 (1990);Liu, W. P., et al., Immunol Lett 93:131 (2004)), not by neutralizing thevirus and preventing infectivity, but by killing infected cells anddisrupting the viral life cycle (Zebedee, S. L., et al., J. Virol.62:2762 (1998); Jegerlehner, A. N., et. al., J Immunol 172:5598 (2004)),which may be by antibody-dependent NK cell activity (Jegerlehner, A. N.,et al., J Immunol 172:5598 (2004)). Composition that include M2eproteins may limit the severity of influenza A disease while allowingthe host immune response to develop adaptive immunity to the dominantneutralizing influenza antigen, HA.

Strategies to manage infection and illness consequent to influenza viralinfection have not changed significantly in the past four decades. Dueto the seasonal nature of influenza disease, the distinct types ofinfluenza virus (A and B) that threaten the human population, and thegenetic instability of each type, it is necessary to reformulate amultivalent vaccine each year, based on epidemiological prediction ofstrains likely to be circulating in the human population in the upcomingflu season. The vaccine is produced from stocks of selected prototypeviral strains grown in embryonated chicken eggs. Thus, the currentstrategy has several limitations that include: (a) dependence onuncertain prediction of circulating strains; (b) dependence on theability to grow the appropriate strains in chicken eggs; (c) theegg-based production system carries risks of product contamination; (d)the product produced in eggs cannot be used in individuals with eggallergies; and (e) a significant risk that the typical multivalentvaccine will not confer protection against a pandemic strain of virus towhich the human population has no pre-existing immunity.

The dominant protective component of the currently-available influenzavaccine is the viral hemagglutinin (HA). A more effective vaccine mayinclude not only strain-specific HA, but cross-protective antigens, suchas M2e and maturational cleavage site. A vaccine production process thatis more reliable, economical, and scaleable than the current egg-basedmethod is also preferable. The compositions, fusion proteins andpolypeptides of the invention provide compositions that include HA, M2e,and maturational cleavage site of influenza proteins, which canstimulate an immune response, specifically, a protective immune responseto several influenza antigens in subject.

The teachings of all patents, published applications and referencescited herein are incorporated by reference in their entirety.

EXEMPLIFICATION Example 1 Design of Portions of a Naturally OccurringHemagglutinin of Influenza A Materials and Methods

Design of the HA 1-1, 1-2 and 1-3 Globular Head Constructs for A/PuertoRico/8/34 (H1N1).

Influenza strain A/Puerto Rico/8/34 (PR8) is a well-characterizedmouse-adapted strain of influenza A virus. The published crystalstructure of the mature PR8HA (SEQ ID NO: 1) (Gamblin et al. 2005.Science 303:1838-1842; PDB accession number 1RU7) was used incombination with the Molecular Modeling Database to determine theboundaries for three PR8 globular head constructs. The complete list ofsolved crystal structures for influenza A hemagglutinin molecules can befound in the Protein Data Bank (PDB) or National Center forBiotechnology Information (NCBI) Structure website.

The three dimensional crystal structure for the PR8HA was viewed usingthe Cn3D program within the NCBI website. The trimeric HA molecule hasthe shape of a mushroom. The globular head refers to the portion of thehemagglutinin molecule at the top that resembles the mushroom head, andthe coiled-coil stalk refers to the bottom part of the molecule thatresembles the stalk of the mushroom (FIG. 1). The globular head containsthe substrate binding site that binds to sialic acid on the cellsurface, and therefore is important for viral entry. In addition, themajority of neutralizing antibodies epitopes are located near and aroundthis receptor binding site, making the globular head a good target forprotective vaccines. The globular head contains most of the HA1 peptideincluding residues numbered from E51 to K327 in PR8 for example.

The monomeric structure of PR8 chain A, which encompasses the globularhead, was used to guide the design of the PR8HA1-1, 1-2 and 1-3constructs (FIG. 1). The HA constructs were designed with domainboundaries within the globular head so that when the molecule isexpressed in a host cell, the encoded protein it may spontaneously foldor refolded in vitro to mimic the native conformation. A structuraldomain (also referred to herein as “domains”) within a protein is anelement of overall structure that is self-stabilizing and often foldsindependently of the rest of the protein chain. Most domains can beclassified into folds. Many domains are not unique to the proteinsproduced by one gene or one gene family but instead appear in a varietyof proteins. Domains often are named and singled out because they playan important role in the biological function of the protein to whichthey belong; for example, the substrate binding domain of hemagglutinincan participate in binding to the substrate. Because domains can beself-stabilizing, domains can be swapped between one protein and anotheror associated to other carrier proteins by genetic engineering to makechimera proteins. A domain may be composed of none, one, or manystructural motifs. In the cases of influenza hemagglutinin constructdesign, many structural motifs are preserved. The boundary selection isguided by known crystal structures. In the case of PR8, residues fromE51 to K327 (SEQ ID NO: 1) fold into a compact structure thatdistinguishes from the rest of the HA molecules, thus is the region offocus to design HA constructs.

Design of the HA 1-1, 1-2 and 1-3 Globular Head Constructs for A/VietNam/1203/2004 (H5N1).

The HA structure used as a reference in the design of the VietNam/1203/2004 (H5N1) globular head constructs is described in Stevens etal, 2006, Science 312:404-410 (MMDB number 38730; PDB accession number2FK0). As described for the PR8 globular head constructs above, thepublished crystal structure of the mature A/Viet Nam/1203/2004 HA (SEQID NO: 2) was used in combination with the Molecular Modeling Databaseto determine the domain boundaries for three Viet Nam globular headconstructs. The same structural criteria for preservation of secondaryand tertiary structure for the structural domain were applied to thedesign of the Viet Nam constructs. While different hemagglutininmolecules can differ in the nature or number of residues that comprisethe hemagglutinin molecule; the overall structure is remarkably similar.Thus, design of the domain boundaries for the Viet Nam globular headconstructs required placement of the boundaries at structurallyequivalent but numerically different positions with in the HA molecule.

Design of the HA 1-1, 1-2 and 1-3 Globular Head Constructs forA/Indonesia/5/2005 (H5N1).

The crystal structure for the Indonesia HA has not been solved. Ingeneral, when the crystal structure for a given HA molecule has not beensolved, the available structure with the highest sequence identity canbe used to guide the construct design. In the case of the design of theIndonesia globular head construct, the closest structure available isthe A/Viet Nam/1203/2004 (Stevens et al. 2006. Science 312:404-410; MMDBnumber 38730; PDB Accession number 2FK0), which is of the same subtypeas A/Indonesia/5/2005.

To design globular head constructs for this HA, the primary sequence ofthe Indonesia HA (SEQ ID NO: 3) was first aligned with A/VietNam/1203/2004 HA (H5VN; SEQ ID NO: 2) molecule (primary sequenceidentity: 96.13%). The primary sequence alignment for A/VietNam/1203/2004 HA (H5VN; SEQ ID NO: 2) and A/Indonesian/5/2005 (H5IN; SEQID NO: 3) was conducted using CLUSTALW and is shown below, where (*)asterisk=identity, (:) colon=conservative substitution; (.)period=weakly conservative substitution; and (space)=divergentsubstitution.

                                           50 H5INMEKIVLLLAIVSLVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILE H5VNMEKIVLLFAIVSLVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILE*******:******************************************                                          100 H5INKTHNGKLCDLDGVKPLILRDCSVAGWLLGNPMCDEFINVPEWSYIVEKAN H5VNKKHNGKLCDLDGVKPLILRDCSVAGWLLGNPMCDEFINVPEWSYIVEKAN*.************************************************                                          150 H5INPTNDLCYPGSFNDYEELKHLLSRINHFEKIQIIPKSSWSDHEASSGVSSA H5VNPVNDLCYPGDFNDYEELKHLLSRINHFEKIQIIPKSSWSSHEASLGVSSA*.*******.*****************************.**** *****                                          200 H5INCPYLGSPSFFRNVVWLIKKNSTYPTIKKSYNNTNQEDLLVLWGIHHPNDA H5VNCPYQGKSSFFRNVVWLIKKNSTYPTIKRSYNNTNQEDLLVLWGIHHPNDA*** *..********************:**********************                                          250 H5INAEQTRLYQNPTTYISIGTSTLNQRLVPKIATRSKVNGQSGRMEFFWTILK H5VNAEQTKLYQNPTTYISVGTSTLNQRLVPRIATRSKVNGQSGRMEFFWTILK****:**********:***********:**********************                                          300 H5INPNDAINFESNGNFIAPEYAYKIVKKGDSAIMKSELEYGNCNTKCQTPMGA H5VNPNDAINFESNGNFIAPEYAYKIVKKGDSTIMKSELEYGNCNTKCQTPMGA****************************:*********************                                          350 H5ININSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQRESRRKKRGLFG H5VNINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQRERRRKKRGLFG**************************************** *********                                          400 H5INAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNS H5VNAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNS**************************************************                                          450 H5INIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMEN H5VNIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMEN**************************************************                                          500 H5INERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHKCDNECMESIRN H5VNERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHKCDNECMESVRN***********************************************:**                                          550 H5INGTYNYPQYSEEARLKREEISGVKLESIGTYQILSIYSTVASSLALAIMMA H5VNGTYDYPQYSEEARLKREEISGVKLESIGIYQILSIYSTVASSLALAIMVA***:************************ *******************:*                                          568 H5INGLSLWMCSNGSLQCRICI (SEQ ID NO: 3) H5VN GLSLWMCSNGSLQCR--- (SEQ ID NO: 2)***************

Design of the HA 1-1, 1-2 and 1-3 Globular Head Constructs for A/NewCaledonia/20/1999 (H1N1).

The crystal structure for the New Caledonia HA has not been solved. Todesign the globular head constructs for this HA, the primary sequence ofthe New Caledonia HA (H1NC; SEQ ID NO: 4) was first aligned with twoclosely related HA molecules of the same subtype (primary sequencehomology >85%) for which the structures have been resolved, specificallythe H1N1 1918 virus (same as A/South Carolina/1/18) (SEQ ID NO: 5) andthe A/Puerto Rico/8/34 virus (H1PR8; SEQ ID NO: 1) (Gamblin, et al.,Science 303, 1838-42 (2004). A/South Carolina/1/18: MMDB number 26943,PDB accession number: 1RUZ and PR8, MMDB number: 26941 and PDB accessionnumber: 1RU7). The primary sequence alignment was conducted usingCLUSTALW and is shown below, where (*) asterisk=identity, (:)colon=conservative substitution; (.) period=weakly conservativesubstitution; and (space)=divergent substitution.

                                                50 H1NCMKAKLLVLLCTFTATYADTICIGYHANNSTDTVDTVLEKNVTVTHSVNLL H1PR8MKANLLVLLSALAAADADTICIGYHANNSTDTVDTVLEKNVTVTHSVNLL ***:*****.:::*: **********************************                                               100 H1NCEDSHNGKLCLLKGIAPLQLGNCSVAGWILGNPECELLISKESWSYIVETP H1PR8EDSHNGKLCRLKGIAPLQLGKCNIAGWLLGNPECDPLLPVRSWSYIVETP ********* **********:*.:***:******: *:. .*********                                               150 H1NCNPENGTCYPGYFADYEELREQLSSVSSFERFEIFPKESSWPNHTVTGVSA H1PR8NSENGICYPGDFIDYEELREQLSSVSSFERFEIFPKESSWPNHNTNGVTA *.*** **** * ******************************...**:*                                               200 H1NCSCSHNGKSSFYRNLLWLTGKNGLYPNLSKSYVNNKEKEVLVLWGVHHPPN H1PR8ACSHEGKSSFYRNLLWLTEKEGSYPKLKNSYVNKKGKEVLVLWGIHHPPN :***:************* *:* **:*.:****:* ********:*****                                               250 H1NCIGNQRALYHTENAYVSVVSSHYSRRFTPEIAKRPKVRDQEGRINYYWTLL H1PR8SKEQQNIYQNENAYVSVVTSNYNRRFTPEIAERPKVRDQAGRMNYYWTLL   :*: :*:.********:*:*.********:******* **:*******                                               300 H1NCEPGDTIIFEANGNLIAPWYAFALSRGFGSGIITSNAPMDECDAKCQTPQG H1PR8KPGDTIIFEANGNLIAPMYAFALSRGFGSGIITSNASMHECNTKCQTPLG :**************** ******************.*.**::***** *                                               350 H1NCAINSSLPFQNVHPVTIGECPKYVRSAKLRMVTGLRNIPSIQSRGLFGAIA H1PR8AINSSLPYQNIHPVTIGECPKYVRSAKLRMVTGLRNTPSIQSRGLFGAIA *******:**:************************* *************                                               400 H1NCGFIEGGWTGMVDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVIE H1PR8GFIEGGWTGMIDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNTVIE **********:***********************************:***                                               450 H1NCKMNTQFTAVGKEFNKLERRMENLNKKVDDGFLDIWTYNAELLVLLENERT H1PR8KMNIQFTAVGKEFNKLEKRMENLNKKVDDGFLDIWTYNAELLVLLENERT *** *************:********************************                                               500 H1NCLDFHDSNVKNLYEKVKSQLKNNAKEIGNGC-------------------- H1PR8LDFHDSNVKNLYEKVKSQLKNNAKEIGNGCFEFYHKCDNECMESVRNGTY ******************************                                               550 H1NC-------------------------------------------------- H1PR8DYPKYSEESKLNREKVDGVKLESMGIYQILAIYSTVASSLVLLVSLGAIS                                               565 H1NC---------------  (SEQ ID NO: 4) H1PR8 FWMCSNGSLQCRICI  (SEQ ID NO: 1)

Design of the HA 1-1, 1-2 and 1-3 Globular Head Constructs forA/Wisconsin/67/2005 (H3N2).

The crystal structure for the Wisconsin HA has not been solved. Todesign the globular head constructs for this HA, the primary sequence ofthe Wisconsin HA (H3W is; SEQ ID NO: 6) was first aligned with a closelyrelated reference HA molecule of the same subtype (primary sequenceidentity: 81.16%) for which the structure had been resolved,specifically influenza A/X31 subtype H3N2 (H3×31; SEQ ID NO: 7) (PDBaccession number: 1VIU). The Wisconsin HA sequence was aligned with theX31 HA using CLUSTAL W and is shown below, where (*) asterisk=identity,(:) colon=conservative substitution; (.) period=weakly conservativesubstitution; and (space)=divergent substitution. Each amino acid in X31was found to have a corresponding match in A/Wisconsin/67/2005 sequence.The domain boundaries the X31 structure were then used to identify thedomain boundaries in the A/Wisconsin/67/2005.

                                                50 H3WisQKLPGNDNSTATLCLGHHAVPNGTIVKTITNDQIEVTNATELVQSSSTGG H3X31QDLPGNDNSTATLCLGHHAVPNGTLVKTITDDQIEVTNATELVQSSSTGK*.**********************:*****:******************                                               100 H3WisICDSPHQILDGENCTLIDALLGDPQCDGFQNKKWDLFVERSKAYSNCYPY H3X31ICNNPHRILDGIDCTLIDALLGDPHCDVFQNETWDLFVERSKAFSNCYPY**:.**:**** :***********:** ***:.**********:******                                               150 H3WisDVPDYASLRSLVASSGTLEFNDESFNWTGVTQNGTSSACKRRSNNSFFSR H3X31DVPDYASLRSLVASSGTLEFITEGFTWTGVIQNGGSNACKRGPGSGFFSR********************  *.*.**** *** *.**** ....****                                               200 H3WisLNWLTHLKFKYPALNVTMPNNEKFDKLYIWGVHHPGTDNDQIFLHAQASG H3X31LNWLTKSGSTYPVLNVTMPNNDNFDKLYIWGIHHPSTNQEQTSLYVQASG*****:   .**.********::********:***.*:::*  *:.****                                               250 H3WisRITVSTKRSQQTVIPNIGSRPRIRNIPSRISIYWTIVKPGDILLINSTGN H3X31RVTVSTRRSQQTIIPNIGSRPWVRGLSSRISIYWTIVKPGDVLVINSNGN*:****:*****:******** :*.:.**************:*:***.**                                               300 H3WisLIAPRGYFKIRSGKSSIMRSDAPIGKCNSECITPNGSIPNDKPFQNVNRI H3X31LIAPRGYFKMRTGKSSIMRSDAPIDTCISECITPNGSIPNDKPFQNVNKI*********:*:************..* ********************:*                                               329 H3WisTYGACPRYVKQNTLKLATGMRNVPEKQTR  (SEQ ID NO: 6) H3X31TYGACPKYVKQNTLKLATGMRNVPEKQT-  (SEQ ID NO: 7)******:*********************

Results

Depiction of Domain Boundaries for PR8HA Constructs.

The selected boundary domains are highlighted in the sequence below (SEQID NO: 1) as follows: HA1-1 boundaries are single-underlined (S53—R324of SEQ ID NO: 1); HA1-2 boundaries are double-underlined (K62-5284 ofSEQ ID NO: 1); HA1-3 boundaries are bold-underlined (N101-G276 of SEQ IDNO: 1). Detailed descriptions of each subunit design and boundarydomains are given below.

(SEQ ID NO: 1) 60MKANLLVLLSALAAADADTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDSHNGKLCR 120

180 QLSSVSSFERFEIFPKESSWPNHNTNGVTAACSHEGKSSFYRNLLWLTEKEGSYPKLKNS 240YVNKKGKEVLVLWGIHHPPNSKEQQNLYQNENAYVSVVTSNYNRRFTPEIAERPKVRDQA 300

360 AINSSLPYQNIHPVTIGECPKYVRSAKLRMVTGLRNIPSIQSRGLFGAIAGFIEGGWTGM 420IDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNTVIEKMNIQFTAVGKEFNKLEKRM 480ENLNKKVDDGFLDIWTYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEIGNGC 540FEFYHKCDNECMESVRNGTYDYPKYSEESKLNREKVDGVKLESMGIYQILAIYSTVASSL 565VLLVSLGAISFWMCSNGSLQCRICI

PR/8 HA1-1 Construct (SEQ ID NO: 8).

For this construct, four (4) conserved disulfide bonds were preserved bymaking the amino-terminal truncation before the Cysteine at position 59of SEQ ID NO: 1 and the carboxyl-terminal truncation after the Cysteineat position 319 of SEQ ID NO: 1. The preserved disulfide bonds arelisted as the following: C59-C291; C72-C84; C107-C152 and C295-C319 ofSEQ ID NO: 1. Using the crystal structure cited above and the MolecularModeling Database (MMDB) to identify tertiary and secondary structure,the amino terminal truncation was placed at the Serine at position 53 ofSEQ ID NO: 1, which is immediately adjacent to the coil turn formed byresidues 50-52 of SEQ ID NO: 1 (LED). The truncation completelyeliminated the preceding β-strand formed by residues 44-49 of SEQ ID NO:1 [THSVNL (SEQ ID NO: 214)]. The strand spanning residues 53-58 of SEQID NO: 1 [SHNGKL (SEQ ID NO: 215)] and the entire β-strand associatedwith residues 59-62 of SEQ ID NO: 1 [CRLK (SEQ ID NO: 216)] werepreserved. Although in the structure residues 53-58 of SEQ ID NO: 1[SHNGKL (SEQ ID NO: 215)] are defined as a random coil, these residuesmimic a β-strand and thus serve to further stabilize the β-pleatedsheets defined by residues 307-310 of SEQ ID NO: 1 [PYQN, SEQ ID NO:217] and residues 320-323 of SEQ ID NO: 1 [PKYV, SEQ ID NO: 218]. Thecarboxyl-terminal truncation was made in the random coil sequencespanning residues 324-327 of SEQ ID NO: 1 [RSAK, SEQ ID NO: 219] at theArginine at position 324 of SEQ ID NO: 1 such that the carboxyl-terminaltail which interacts with HA2 was completely eliminated.

PR/8 HA1-2 Construct (SEQ ID NO: 9).

For this construct two (2) conserved disulfide bonds (C72-C84 andC107-C152 of SEQ ID NO: 1) were preserved by making the amino-terminaltruncation before the Cysteine at position 72 of SEQ ID NO: 1 and thecarboxyl-terminal truncation after the Cysteine at position 152 of SEQID NO: 1. Using the crystal structure cited above and the MMD databaseto identify tertiary and secondary structure, the amino-terminal and thecarboxyl-terminal truncations were placed in the loops [KGI, residues62-64 of SEQ ID NO: 1 and NASMHE (SEQ ID NO: 220), residues 285-290, ofSEQ ID NO: 1] that connect two distinct sets of β-pleated sheets.Truncation of the molecule within these loops preserves the surroundingsecondary structure which in turn preserves the tertiary structure ofthe globular head.

The amino-terminal truncation was made at the Lysine at position 62 ofSEQ ID NO: 1, such that the β-strand comprising APLQLG (SEQ ID NO: 221)at residues 65-70 of SEQ ID NO: 1 of the membrane-distal set ofβ-strands remained wholly intact, while the preceding β-strand [CRLK,SEQ ID NO: 216, residues 59-62 of SEQ ID NO: 1] that is moremembrane-proximal was largely eliminated. The carboxyl-terminaltruncation was made at the Serine at position 284 of SEQ ID NO: 1 suchthat the β-strand [GIITS, SEQ ID NO: 222, residues 280-284 of SEQ ID NO:1] of the distal set of β-strands remains wholly or largely intact andthe subsequent β-strand [CNTK, SEQ ID NO: 223, residues 291-294 of SEQID NO: 1] of the proximal set of β-stands is wholly or largelyeliminated. Preservation of the distal set of β-strands was the maindeterminant in selecting the domain boundaries for the HA1-2 construct.The distal β-pleated sheet, which is comprised of β-strands of APLQLG(SEQ ID NO: 221) [residues 65-70 of SEQ ID NO: 1], SYIVET (SEQ ID NO:224) [residues 94-99 of SEQ ID NO: 1], and GIITS (SEQ ID NO: 222)[residues 280-284 of SEQ ID NO: 1], serves as the stabilizing secondarystructural element that ensures a compact domain structure.

PR/8 HA1-3 Construct (SEQ ID NO: 10).

For this construct one (1) conserved disulfide bond (C107-C152 of SEQ IDNO: 1) was preserved by making the amino-terminal truncation before theCysteine at position 107 of SEQ ID NO: 1 and the carboxyl terminaltruncation after the Cysteine at position 152 of SEQ ID NO: 1. Using thecrystal structure cited above and the MMDB database to identify tertiaryand secondary structure the amino-terminal truncation was made at theAsparagine at position 101 of SEQ ID NO: 1 such that the precedingα-helix [NIAGWLLG, SEQ ID NO: 225, residues 73-80 of SEQ ID NO: 1] andβ-strand [SYIVET, SEQ ID NO: 224, residues 94-99 of SEQ ID NO: 1] werecompletely eliminated while the subsequent β-stand [PGDFI, SEQ ID NO:226, residues 109-113 of SEQ ID NO: 1] was preserved along with thestrand NSENGICY, SEQ ID NO: 227 [residues 101-108 of SEQ ID NO: 1] thatincludes the conserved Cysteine at position 107 of SEQ ID NO: 1.

The carboxyl-terminal truncation was made near the end of the β-strand[AFALSRGF, SEQ ID NO: 228, residues 270-277 of SEQ ID NO: 1]. The lastamino acid of this strand, position 277 of SEQ ID NO: 1, is aPhenylalanine, which was eliminated so as not to expose this hydrophobicresidue. The carboxyl-terminal truncation was made at the Glycine atposition 276 of SEQ ID NO: 1 which is in between two secondarystructures. Truncation at this position keeps the preceding β-strand[AFALSRG, SEQ ID NO: 229, residues 270-276 of SEQ ID NO: 1] largelyintact and completely eliminates the subsequent β-strand [GIITS, SEQ IDNO: 222, residues 280-284 of SEQ ID NO: 1].

Depiction of Domain Boundaries for VN04 HA Construct.

The selected boundary domains are highlighted in the sequence below (SEQID NO: 2) as follows: HA1-1 boundaries are single-underlined (E50-K323of SEQ ID NO: 2); HA1-2 boundaries are double-underlined (G62-E284 ofSEQ ID NO: 2); HA1-3 boundaries are bold-underlined (N103-G276 of SEQ IDNO: 2). Detailed descriptions of each subunit design and boundarydomains are given below.

(SEQ ID NO: 2) 60MEKIVLLFAIVSLVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKKHNGKLCDL 120

180 LSRINHFEKIQIIPKSSWSSHEASLGVSSACPYQGKSSFFRNVVWLIKKNSTYPTIKRSY 240NNTNQEDLLVLWGIHHPNDAAEQTKLYQNPTTYISVGTSTLNQRLVPRIATRSKVNGQSG 300

360 INSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQRERRRKKRGLFGAIAGFIEGGW 420QGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLE 480RRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELG 540NGCFEFYHKCDNECMESVRNGTYDYPQYSEEARLKREEISGVKLESIGIYQILSIYSTVA 565SSLALAIMVAGLSLWMCSNGSLQCR

VN04 HA1-1 Construct (SEQ ID NO: 11).

For this construct four (4) conserved disulfide bonds were preserved bymaking the amino terminal truncation before the Cysteine at position 58of SEQ ID NO: 2 and the carboxyl-terminal truncation after the Cysteineat position 318 of SEQ ID NO: 2. The preserved disulfide bonds arelisted as the following: C58-C290; C71-C83; C106-C151 and C294-C318 ofSEQ ID NO: 2. Using the crystal structure cited above and the MolecularModeling Database (MMDB) to identify tertiary and secondary structure,the amino terminal truncation was placed at the Glutamic Acid found atposition 50 of SEQ ID NO: 2 such that the preceding β-strand [THAQDI,SEQ ID NO: 230, residues 43-48 of SEQ ID NO: 2] is totally eliminatedand the subsequent strand [EKKHNG, SEQ ID NO: 231, residues 50-55 of SEQID NO: 2] and the β-strand [KLCDLD, SEQ ID NO: 232] formed by residues56-61 of SEQ ID NO: 2 were left wholly intact. Structurally the randompeptide comprising residues 50-55 of SEQ ID NO: 2 [EKKHNG, SEQ ID NO:231] mimics a β-strand that completes the membrane-distal β-pleatedsheet to keep the domain structure intact. The carboxyl-terminaltruncation was made in the loop region near residues 323-326 of SEQ IDNO: 2 [KSNR, SEQ ID NO: 233] that follow the Cysteine at position 318 ofSEQ ID NO: 2. Truncation within this loop preserves secondary structurein that the preceding β-strand formed by residues 319-322 of SEQ ID NO:2 [PKYV, SEQ ID NO: 234] remains wholly intact and the subsequentβ-strand [LVLATG, SEQ ID NO: 235, residues 327-332 of SEQ ID NO: 2] iscompletely eliminated.

VN04 HA1-2 Construct (SEQ ID NO: 12).

For this construct two (2) conserved disulfide bonds (C71-C83 andC106-C151 of SEQ ID NO: 2) were preserved by making the amino-terminaltruncation before the Cysteine at position 71 of SEQ ID NO: 2 and thecarboxyl-terminal truncation after the Cysteine at position 151 of SEQID NO: 2. Using the crystal structure cited above and the MolecularModeling Database (MMDB) to identify tertiary and secondary structure,the amino terminal truncation was placed at the glycine at position 62of SEQ ID NO: 2 such that the subsequent β-strand comprising residues64-68 of SEQ ID NO: 2 [KPLIL, SEQ ID NO: 236] remains wholly intact,while the preceding β-strand [KLCDLD, SEQ ID NO: 232, residues 56-61 ofSEQ ID NO: 2] is completely eliminated.

To preserve secondary structure, the carboxyl-terminal truncation wasmade in the loop region at the Glutamic Acid at position 284 of SEQ IDNO: 2 such that the preceding β-strand [TIMKS, SEQ ID NO: 237, residues279-283 of SEQ ID NO: 2] remained wholly or largely intact and thesubsequent β-strand [YGNCN, SEQ ID NO: 238, residues 287-291 of SEQ IDNO: 2] was completely eliminated.

VN04 HA1-3 Construct (SEQ ID NO: 13).

For this construct one (1) conserved disulfide bond (C106-C151 of SEQ IDNO: 2) was preserved by making the amino-terminal truncation before theCysteine at position 106 of SEQ ID NO: 2 and the carboxyl terminaltruncation after the Cysteine at position 151 of SEQ ID NO: 2.

Using the crystal structure cited above and the Molecular ModelingDatabase (MMDB) to identify tertiary and secondary structure, the aminoterminal truncation was placed at the Asparagine at position 103 of SEQID NO: 2 such that the preceding β-strand [SYIVEK, SEQ ID NO: 239,residues 93-98 of SEQ ID NO: 2] was completely eliminated while thesubsequent β-strand sequence [PGDFN, SEQ ID NO: 240, residues 108-112 ofSEQ ID NO: 2] remained wholly or largely intact. To preserve secondarystructure, the carboxyl-terminal truncation was made in the random coilregion at the Glycine at position 276 of SEQ ID NO: 2 such that thepreceding β-strand formed by residues 102-105 of SEQ ID NO: 2 [KGDS, SEQID NO: 241] remains wholly or partially intact, the preceding β-strand[EYAYKIVK, SEQ ID NO: 242, residues 267-274 of SEQ ID NO: 2] is whollypreserved and the subsequent β-strand [TIMKS, SEQ ID NO: 237, residues279-283 of SEQ ID NO: 2] is completely eliminated.

Depiction of Domain Boundaries for IND05 HA Constructs.

The A/Indonesia/5/2005 construct description is based on sequencealignment in reference to A/Viet Nam/1203/2004 structure. The selectedboundary domains are highlighted in the sequence below (SEQ ID NO: 3) asfollows: HA1-1 boundaries are single-underlined (E50-K323, of SEQ ID NO:3); HA1-2 boundaries are double-underlined (G62-E284 of SEQ ID NO: 3);HA1-3 boundaries are bold-underlined (N103-G276 of SEQ ID NO: 3).Detailed descriptions of each subunit design and boundary domains aregiven below.

(SEQ ID NO: 3) 60MEKIVLLLAIVSLVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCDL 120

180 LSRINHFEKIQIIPKSSWSDHEASSGVSSACPYLGSPSFFRNVVWLIKKNSTYPTIKKSY 240NNTNQEDLLVLWGIHHPNDAAEQTRLYQNPTTYISIGTSTLNQRLVPKIATRSKVNGQSG 300

360 INSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQRESRRKKRGLFGAIAGFIEGGW 420QGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLE 480RRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELG 540NGCFEFYHKCDNECMESIRNGTYNYPQYSEEARLKREEISGVKLESIGTYQILSIYSTVA 560SSLALAIMMAGLSLWMCSNGSLQCRICI

IND05 HA1-1 Construct (SEQ ID NO: 14).

For this construct four (4) conserved disulfide bonds were preserved bymaking the amino-terminal truncation before the Cysteine at position 58of SEQ ID NO: 3 and the carboxyl-terminal truncation after the Cysteineat position 318 of SEQ ID NO: 3. The preserved disulfide bonds arelisted as the following: C58-C291; C71-C83; C106-C151 and C294-C318 ofSEQ ID NO: 3. Using the crystal structure cited above and the MolecularModeling Database (MMDB) to identify tertiary and secondary structure,the amino terminal truncation was placed at the Glutamic Acid found atposition 50 of SEQ ID NO: 3 such that the preceding β-strand [THAQDI,SEQ ID NO: 243, residues 43-48 of SEQ ID NO: 3] was totally eliminatedand the subsequent strand [EKTHNG, SEQ ID NO: 244, residues 50-55 of SEQID NO: 3] and β-strand [KLCDLD, SEQ ID NO: 245] formed by residues 56-61of SEQ ID NO: 3 was left wholly intact. Structurally the random peptidecomprising residues 50-55 of SEQ ID NO: 3 [EKTHNG, SEQ ID NO: 244]mimics a β-strand that completes the membrane-distal β-pleated sheet tokeep the domain structure intact. The carboxyl-terminal truncation wasmade in the loop region near residues 323-326 of SEQ ID NO: 3 [KSNR, SEQID NO: 246] that follow the Cysteine at position 318 of SEQ ID NO: 3.Truncation within this loop preserves secondary structure in that thepreceding β-strand formed by residues 319-322 of SEQ ID NO: 3 [PKYV, SEQID NO: 247] remains wholly intact and the subsequent β-strand [LVLATG,SEQ ID NO: 248, residues 327-332 of SEQ ID NO: 3] is completelyeliminated.

IND05 HA1-2 Construct (SEQ ID NO: 15).

For this construct two (2) conserved disulfide bonds (C71-C83 andC106-C151 of SEQ ID NO: 3 were preserved by making the amino-terminaltruncation before the Cysteine at position 71 of SEQ ID NO: 3 and thecarboxyl-terminal truncation after the Cysteine at position 151 of SEQID NO: 3. Using the crystal structure cited above and the MolecularModeling Database (MMDB) to identify tertiary and secondary structure,the amino terminal truncation was placed at the Glycine at position 62of SEQ ID NO: 3 such that the subsequent β-strand comprising residues64-68 of SEQ ID NO: 3 [KPLIL, SEQ ID NO: 249] remained wholly intact,while the preceding β-strand [KLCDLD, SEQ ID NO: 245, residues 56-61 ofSEQ ID NO: 3] was completely eliminated. To preserve secondarystructure, the carboxyl terminal truncation was made in the loop regionat the Glutamic Acid at position 284 of SEQ ID NO: 3 in the sequence[ELE, residues 284-286 of SEQ ID NO: 3] such that the preceding β-strand[AIMKS, SEQ ID NO: 250, residues 279-283 of SEQ ID NO: 3] remainedwholly or largely intact and the subsequent β-strand [YGNCN, SEQ ID NO:251, residues 287-291 of SEQ ID NO: 3] was completely eliminated.

IND05 HA1-3 Construct (SEQ ID NO: 16).

For this construct one (1) conserved disulfide bond (C106-C151 of SEQ IDNO: 3 was preserved by making the amino-terminal truncation before theCysteine at position 106 of SEQ ID NO: 3 and the carboxyl-terminaltruncation after the Cysteine at position 151 of SEQ ID NO: 3. Using thecrystal structure cited above and the Molecular Modeling Database (MMDB)to identify tertiary and secondary structure, the amino terminaltruncation was placed at the asparagine at position 103 of SEQ ID NO: 3such that the preceding β-strand [SYIVEK, SEQ ID NO: 252, residues 93-98of SEQ ID NO: 3] was completely eliminated while the β-strand sequence[PGSFN, SEQ ID NO: 253, residues 108-112 of SEQ ID NO: 3] remainedwholly or largely intact. To preserve secondary structure, thecarboxyl-terminal truncation was made in the random coil region at theGlycine found at position 276 of SEQ ID NO: 3 such that the precedingβ-strand [EYAYKIVK, SEQ ID NO: 254, residues 267-274 of SEQ ID NO: 3]was wholly preserved and the subsequent β-strand [AIMKS, SEQ ID NO: 250,residues 279-283 of SEQ ID NO: 3] was completely eliminated.

Depiction of Domain Boundaries for New Caledonia HA Constructs.

A/New Caledonia/20/199 construct description is based on sequencealignment in reference of A/Puerto Rico/8/34 structure. The selectedboundary domains are highlighted in the sequence below (SEQ ID NO: 4) asfollows: HA1-1 boundaries are single-underlined (S53-R324 of SEQ ID NO:4); HA1-2 boundaries are double-underlined (K62-5284 of SEQ ID NO: 4);HA1-3 boundaries are bold-underlined (N101-G276 of SEQ ID NO: 4).Detailed descriptions of each subunit design and boundary domains aregiven below.

(SEQ ID NO: 4) 60MKAKLLVLLCTFTATYADTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDSHNGKLCL 120

180 QLSSVSSFERFEIFPKESSWPNHTVTGVSASCSHNGKSSFYRNLLWLTGKNGLYPNLSKS 240YVNNKEKEVLVLWGVHHPPNIGNQRALYHTENAYVSVVSSHYSRRFTPEIAKRPKVRDQE 300

360 AINSSLPFQNVHPVTIGECPKYVRSAKLRMVTGLRNIPSIQSRGLFGAIAGFIEGGWTGM 420VDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVIEKMNTQFTAVGKEFNKLERRM 480ENLNKKVDDGFLDIWTYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEIGNGC 540FEFYHKCNNECMESVKNGTYDYPKYSEESKLNREKIDGVKLESMGVYQILAIYSTVASSL 565VLLVSLGAISFWMCSNGSLQCRICI

New Caledonia HA1-1 Construct Design (SEQ ID NO: 17).

For this construct, four (4) conserved disulfide bonds were preserved bymaking the amino terminal truncation before the Cysteine at position 59of SEQ ID NO: 4 and the carboxyl-terminal truncation after the Cysteineat position 319 of SEQ ID NO: 4. The preserved disulfide bonds arelisted as the following: C59-C291; C72-C84; C107-C152 and C295-C319 ofSEQ ID NO: 4. Using the reference crystal structures and the MMDB tocheck secondary structures the amino terminal truncation was made at theSerine at position 53 of SEQ ID NO: 4 such that the preceding β-strand[THSVNLLED, SEQ ID NO: 255, residues 44-52 of SEQ ID NO: 4] was totallyeliminated and the subsequent β-strands SHNGKL, SEQ ID NO: 256 [residues53-58 of SEQ ID NO: 4] and APLQLG, SEQ ID NO: 257 [residues 65-70, ofSEQ ID NO: 4] were left wholly intact. The amino-terminal truncation inthe random coil sequence [SHNGKL, SEQ ID NO: 256, residues 53-58 of SEQID NO: 4] left the secondary structure largely intact because it mimicsa β-strand that leaves the β-pleated sheet intact. The carboxyl-terminaltruncation was made at the Arginine at position 324 of SEQ ID NO: 4 inthe random coil sequence [RSAK, SEQ ID NO: 258, residues 324-327 of SEQID NO: 4] following the Cysteine at position 319 of SEQ ID NO: 4. Thetruncation in this random coil preserves secondary structure by leavingthe preceding β-strand [PKYV, SEQ ID NO: 259, residues 320-323 of SEQ IDNO: 4] wholly intact while the subsequent β-strand [LRMVTG, SEQ ID NO:260, residues 328-333 of SEQ ID NO: 4] is completely eliminated.

New Caledonia HA1-2 Construct Design (SEQ ID NO: 18).

For this construct two (2) conserved disulfide bonds (C72-C84 andC107-C152 of SEQ ID NO: 4) were preserved by making the amino-terminaltruncation before the Cysteine at position 72 of SEQ ID NO: 4 and thecarboxyl-terminal truncation after the Cysteine at position 152 of SEQID NO: 4. Using the reference crystal structures and the MMDB to checksecondary structures the amino-terminal truncation was made at theLysine at position 62 of SEQ ID NO: 4 such that the random coil sequencecomprising [SHNGKLCLLKGI, SEQ ID NO: 261, residues 53-64 of SEQ ID NO:4] was partially or largely removed, the preceding β-strand [THSVNLLED,SEQ ID NO: 255, residues 44-52 of SEQ ID NO: 4] was completelyeliminated and the subsequent β-strand [APLQLG, SEQ ID NO: 257, residues65-70 of SEQ ID NO: 4] remained wholly intact. The carboxyl-terminaltruncation was made at the Serine at position 284 of SEQ ID NO: 4 suchthat the preceding β-strand [SGIITS, SEQ ID NO: 262, residues 279-284 ofSEQ ID NO: 4] remained wholly intact and the subsequent random coil[NAPMDECDA, SEQ ID NO: 263, residues 285-293 of SEQ ID NO: 4] was whollyor largely eliminated.

New Caledonia HA1-3 Construct Design (SEQ ID NO: 19).

For this construct one (1) conserved disulfide bond (C107-C152 of SEQ IDNO: 4 was preserved by making the amino-terminal truncation before theCysteine at position 107 of SEQ ID NO: 4 and the carboxyl terminaltruncation after C152 of SEQ ID NO: 4. Using the reference crystalstructures and the MMDB to check secondary structures the amino-terminaltruncation was made at the Asparagine at position 101 of SEQ ID NO: 4such that the receding β-strand [SYIVET, SEQ ID NO: 264, residues 94-99of SEQ ID NO: 4] was completely eliminated and the subsequent β-strand[PGYFA, SEQ ID NO: 265, residues 109-113 of SEQ ID NO: 4] remainedwholly intact. To preserve the secondary structure the carboxyl-terminaltruncation was made at the Glycine at position 276 of SEQ ID NO: 4 suchthat the β-strand sequence [YAFALSRGF, SEQ ID NO: 266, residues 269-277of SEQ ID NO: 4] remained largely intact, while the subsequent β-strandsequence [SGIITS, SEQ ID NO: 262, residues 279-284 of SEQ ID NO: 4] waseliminated.

Depiction of Domain Boundaries for A/Wisconsin/67/2005 HA Constructs.

A/Wisconsin/67/2005 construct description is based on sequence alignmentin reference of A/X31 structure. The selected boundary domains arehighlighted in the sequence below (SEQ ID NO: 6) as follows: HA1-1boundaries are single-underlined (Q44-K310 of SEQ ID NO: 6); HA1-2boundaries are double-underlined (S54-D271 of SEQ ID NO: 6); HA1-3boundaries are bold-underlined (S95-G263 of SEQ ID NO: 6). Detaileddescriptions of each subunit design and boundary domains are givenbelow.

(SEQ ID NO: 6) 60QKLPGNDNSTATLCLGHHAVPNGTIVKTITNDQIEVTNATELVQSSSTGGICDSPHQILD 120

180 NDESFNWTGVTQNGTSSACKRRSNNSFFSRLNWLTHLKFKYPALNVTMPNNEKFDKLYIW 240GVHHPGTDNDQIFLHAQASGRITVSTKRSQQTVIPNIGSRPRIRNIPSRISIYWTIVKPG 300

329 TYGACPRYVKQNTLKLATGMRNVPEKQTR

A/Wisconsin/67/2005 HA1-1 Construct Design (SEQ ID NO: 20).

For this construct, four (4) conserved disulfide bonds were preserved bymaking the amino terminal truncation before the Cysteine at position 52of SEQ ID NO: 6 and the carboxyl-terminal truncation after the Cysteineat position 305 of SEQ ID NO: 6. The preserved disulfide bonds arelisted as the following: C52-C277; C64-C76; C97-C139 and C281-C305 ofSEQ ID NO: 6. Using the reference crystal structures and the MMDB tocheck secondary structures the amino terminal truncation was made at theGlutamine at position 44 of SEQ ID NO: 6, right after the tight turnformed by residues 42 and 43 of SEQ ID NO: 6 [LV], such that thepreceding β-strand [TNATE, SEQ ID NO: 267, residues 37-41 of SEQ ID NO:6] was totally eliminated while the entire short β-strand formed byresidues QSS [44-46 of SEQ ID NO: 6] was preserved. Thecarboxyl-terminal truncation was made in the random coil sequence[PRYVK, SEQ ID NO: 268, residues 306-310 of SEQ ID NO: 6] following theCysteine at position 305 of SEQ ID NO: 6 such that the carboxyl-terminaltail which interacts with HA2 was completely eliminated. Although thecarboxyl-terminal sequence [PRYVK, SEQ ID NO:268, residues 306-310 ofSEQ ID NO: 6)] is defined as a random coil, structurally it serves as anadditional β-strand that further stabilizes the β-pleated sheets definedby residues 44-46 of SEQ ID NO: 6 [QSS] and residues 293-297 of SEQ IDNO: 6 [PFQNV, SEQ ID NO: 269]. This set of β-pleated encompasses theamino-terminus and carboxy-terminus within one stable secondarystructure element to ensure a compact domain structure.

A/Wisconsin/67/2005 HA1-2 Construct Design (SEQ ID NO: 21).

For this construct two (2) conserved disulfide bonds (C64-C76 andC97-C139 of SEQ ID NO: 6) were preserved by making the amino-terminaltruncation before the Cysteine at position 64 of SEQ ID NO: 6 and thecarboxyl-terminal truncation after the Cysteine at position 139 of SEQID NO: 6. Using the reference crystal structures and the MMDB to checksecondary structures the amino-terminal truncation was made in the loopsthat connect two distinct sets of β-pleated sheets. The amino-terminaltruncation was made at the Serine at position 54 of SEQ ID NO: 6, suchthat the β-strand comprising residues 57-62 of SEQ ID NO: 6 [QILDGE, SEQID NO: 270] of the membrane-distal set of β-strands remained whollyintact, while the membrane-proximal β-strand [GGICD, SEQ ID NO: 271,residues 49-53 of SEQ ID NO: 6] was completely eliminated. Thecarboxyl-terminal truncation was made at the Aspartic Acid at position271 of SEQ ID NO: 6. Truncation at this position preserved secondarystructure such that the membrane-distal β-strand [SSIMRS, SEQ ID NO:272, residues 265-270 of SEQ ID NO: 6] remained intact while themembrane-proximal β-strand [APIGK, SEQ ID NO: 273, residues 272-276 ofSEQ ID NO: 6] was eliminated. The membrane-distal β-pleated sheetscomprising β-strands QILDGE (SEQ ID NO: 270), [residues 57-62 of SEQ IDNO: 6] LFVER (SEQ ID NO: 274) [residues 86-90 of SEQ ID NO: 6], andSSIMRS, (SEQ ID. NO: 272), [residues 265-270 of SEQ ID NO: 6] serve asthe stabilizing secondary structure element that ensures a compactdomain structure.

A/Wisconsin/67/2005 HA1-3 Construct Design (SEQ ID NO: 22).

For this construct one (1) conserved disulfide bond (C97-C139 of SEQ IDNO: 6) was preserved by making the amino-terminal truncation before theCysteine at position 97 of SEQ ID NO: 6 and the carboxy-terminaltruncation after the Cysteine at position 139 of SEQ ID NO: 6. Using thereference crystal structure and the MMDB to check secondary structuresthe amino-terminal truncation was made at the Serine at position 95 ofSEQ ID NO: 6 such that the preceding α-helix [TLIDALLG, SEQ ID NO: 275,residues 65-72 of SEQ ID NO: 6] was completely eliminated while theSerine at position 95 of SEQ ID NO: 6 and Asparagine at position 96 ofSEQ ID NO: 6 were preserved along with the conserved Cysteine atposition 97 of SEQ ID NO: 6. The carboxyl-terminal truncation was madein the random coil sequence at about the Glycine at position 263 of SEQID NO: 6 such that the random coil sequence containing residues 261-263of SEQ ID NO: 6 [RSG] was kept wholly or partially intact, while thepreceding β-strand [RGYFKI, SEQ ID NO: 276, residues 255-260 of SEQ IDNO: 6] was wholly preserved and the subsequent β-strand [SSIMRS, SEQ IDNO: 272, residues 265-270 of SEQ ID NO: 6] was completely eliminated.

Discussion

Parts of the globular head domain of the influenza virus hemagglutinin(HA) protein may generate neutralizing antibodies (Brand and Skehel,1972; Eckert, 1973; Jackson et al., 1979; Russ et al., 1981) and thediscrete, globular structure of this domain was thought to constitute aprotein fragment which could be expressed in bacteria and refolded, orexpressed and secreted from a eukaryotic host, thereby avoiding theexpression and purification problems which have been encounteredproducing the full-length HA protein.

The largest HA construct (also referred to herein as “HA fragment”)fused to STF2, HA1-1 (SEQ ID NO: 8, 11, 14, 17, 20), encompasses most ofor likely the entire globular region of the naturally occurring (wildtype) HA1 head domain. A naturally occurring fragment of HA1 verysimilar to the HA1-1 construct is released from the virus on limitedproteolysis (Bizeband et al., 1995). Thus, structural analysis andexperimental data suggest that the HA1-1 construct should foldefficiently, whether produced in bacteria or eukaryotes, and maintain astable native-like structure. Similarly, it is expected that the entireHA0s, the external portion of HA molecule outside the viral membrane(SEQ ID NO: 23) consisting of HA1 and HA2 but excluding the signalpeptide (17 aa), internal peptide within the viral membrane (12 aa) andtransmembrane domain (24 aa) should attain its native conformation whenexpressed in a eukaryotic host or can be refolded into a native state invitro when expressed in bacteria. The two smaller HA fragments (HA1-2(SEQ ID NO: 9, 12, 15, 18, 21) and HA1-3 (SEQ ID NO: 10, 13, 16, 19,22)) truncate the region of the head domain distal to the receptorbinding site in a manner which may either destabilize the resultingprotein fragment (HA1-3) or expose the side chains of a few largehydrophobic residues (HA1-2). For this reason, experimental mutationswere introduced into the HA1-2 and HA1-3 constructs, denoted as HA1-2mut(SEQ ID NO: 24, 26, 28, 30, 32) and HA1-3mut (SEQ ID NO: 25, 27, 29, 31,33). The substitutions made in the HA1-3mut (SEQ ID NO: 25, 27, 29, 31,33) construct introduce oppositely charged residues on the oppositestrands which are left exposed by the HA1-3 truncations. “HA mut,” asused herein, means an amino acid in the native or naturally occurring HAhas been substituted with an amino acid that does not occur in thenative or naturally occurring HA. Substituted residues may form a saltbridge and stabilize a structure which may otherwise fold poorly, or notat all. For example, in PR8, G105 was replaced by glutamate and Y115 wassubstituted with lysine (SEQ ID NO: 25). Negatively charged glutamateinteracted with positively charged lysine residue so that N-terminalpeptide 1-8 of SEQ ID NO: 25 [NSENEICY, SEQ ID NO: 277] was stabilizedby charge-charge interaction with Lysine 15 located at the N-terminus ofthe short helix 15-22 of SEQ ID NO: 25 [KEELREQL, SEQ ID NO: 278] in thecenter of the molecule. For the HA1-2 construct, the truncations canlead to the exposure of hydrophobic side chains which would not beexpected to impair expression of the protein. However, the exposedresidues may lead to aggregation of expressed protein or instability ofexpressed protein. To avoid aggregation and enhance the stability of theexpressed molecule the few large hydrophobic residues that becomeexposed were substituted with more neutral amino acids to generateHA1-2mut (SEQ ID NO: 24, 26, 28, 30, 32). These substitutions may resultin a protein which is more amenable to a robust manufacturing processand/or long term stability.

Example 2 Design of Portions of a Naturally Occurring Hemagglutinin ofInfluenza B Materials and Methods

Design of the HA1-1, 1-2 and 1-3 Globular Head Constructs for B/Lee/40.

Structural considerations in the design of an influenza B HA vaccine aresimilar to those for influenza A, i.e., the domain boundary of theglobular head of HA must be identified so that the flagellin-HA fusionprotein can fold correctly or be refolded correctly to exposeappropriate antigenic epitopes. Unlike influenza A, well-defined X-raycrystallographic structures are not available for influenza B HA, thusit is more difficult to unambiguously define the domain boundary ofglobular head. Therefore the influenza B HA model must be predictedbased on bioinformatic and structural models (Tung et al. 2004. J. Gen.Virl. 85, 3249-3259). These investigators used a “knowledge-based”approach which depends on a high degree of sequence homology between theknown structure from the protein data bank and the target unknownstructure. In general, this approach benefits most from at least 35%sequence identity between the known and target proteins.

In the case of influenza B, the closest models come from A/Swine/HongKong/9/98 (SEQ ID NO: 34) (24% identity, PDB accession code 1JSD) andA/Aichi/2/68 (SEQ ID NO: 35) (21% identity, PDB accession code 1HGF).Although the sequence identities between the target model B/Lee/40 HA(SEQ ID NO: 36) and known template models are substantially below thedesired minimum of 35%, the close similarity of the functions andtertiary folds of influenza A HA proteins in spite of their sequencedivergence (H1, H3, H5 and H9 share only 18% sequence identity) suggesta possibly successful prediction of influenza B HA structure using theinfluenza A HA model. Moreover, influenza C HEF(Hemagglutinin-Esterase-Fusion) protein folds similarly to influenza AHA structure despite even lower sequence identity than any of the A/Bcomparison.

Since the crystal structure of influenza C HEF is known (PDB accessionnumber 1FLC, MMDB accession number 12663), Tung et al included theknowledge of the structure similarity between C HEF and the knowninfluenza A HA proteins to predict influenza B HA structure. Tung et alfirst aligned the HEF sequence from C/Johannesburg/1/66 (SEQ ID NO: 37)with one sequence from each of the 15 HA subtypes of influenza A virus(http://flu.lanl.gov) using CLUSTALW (Thompson et al. 1994. NucleicAcids Res 22, 4673-4680) and compiled a profile based onstructure-informed alignment.

The conserved secondary structural features were captured and assigned,as well as the variations among the types and subtypes by year and hostspecies. They then further aligned the augmented A/C profile to analignment of influenza B HA sequences, including B/Lee/40. The homologymodeling techniques of Tung (1999) was used to construct B/Lee/40 model.

Briefly, they first matched the main-chain structures of the target tothose of the template in the aligned regions. Insertions in the targetrelative to the template were treated as loops with known end-structure.Stretching the predicted structure accommodated insertions in thetemplate relative to the target. The mainchain structures of the loopswere modeled by using an efficient Monte Carlo loop-sampling method(Tung, 1997; Ryu et al., 1998). Once the main-chain structure wasmodeled, the side-chain atoms were attached. As the head of the HAmolecule is compact, limited space is available to place the side-chainatoms. Hence, in the analysis, side-chain torsional angles wereinitialized to equal or be close to those in the template structure.This consideration is particularly useful in avoiding clashes betweenside chains in the modeled structure. Finally, the all-atom models weresubjected to a short run of energy minimization (1000 cycles) by usingAMBER (Weiner et al., 1986) to relieve unfavourable steric interactionsand to optimize the stereochemistry. The quality of the target model waschecked by PROCHECK (Wilson et al. 1998. J. Mol. Biol. 276, 417-436) andthe functionality was checked by substrate docking to test whether themodel substrate binding site can accommodate the natural receptoranalogue sialyllactose as we have seen in the crystal structure of theHA of A/Aichi/2/68 (Weis et al. 1988. Nature 333, 426-431). Thesimulated B/Lee/40 model appears reasonable in a Ramachandran Plot(Wilson et al. 1998. J. Mol. Biol. 276, 417-436) and shows no stericcrash when docking the sialyl-2-3-lactose molecule into the substratebinding site of the B/Lee/40 model, indicating a correctstereochemistry. Therefore the B/Lee/40 model was used to guide thedesign of influenza B HA subunit vaccines.

Results and Discussion

Depiction of Domain Boundaries for B/Lee/40 HA Constructs.

The selected boundary domains are highlighted in the sequence below asfollows: HA1-1 boundaries are single-underlined (T48-K340 of SEQ ID:36), and HA1-2 boundaries are double-underlined (K60-G299 of SEQ ID:36). Detailed descriptions of each subunit design and boundary domainsare given below.

(SEQ. ID NO: 36)MKAIIVLLMVVTSNADRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTPTKSHFANLK 60GTQTRGKLCPNCFNCTDLDVALGRPKCMGNTPSAKVSILHEVKPATSGCFPIMHDRTKIR 120QLPNLLRGYENIRLSTSNVINTETAPGGPYKVGTSGSCPNVANGNGFFNTMAWVIPKDNN 180KTAINPVTVEVPYICSEGEDQITVWGFHSDDKTQMERLYGDSNPQKFTSSANGVTTHYVS 240QIGGFPNQTEDEGLKQSGRIVVDYMVQKPGKTGTIVYQRGILLPQKVWCASGRSKVIKGS 300LPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLKE 360RGFFGAIAGFLEGGWEGMIAGWHGYTSHGAHGVAVAADLKSTQEAINKITKNLNYLSELE 420VKNLQRLSGAMNELHDEILELDEKVDDLRADTISSQIELAVLLSNEGIINSEDEHLLALE 480RKLKKMLGPSAVEIGNGCFETKHKCNQTCLDRIAAGTFNAGDFSLPTFDSLNITAASLND 540DGLDNHTILLYYSTAASSLAVTLMIAIFIVYMVSRDNVSCSICL 584

The coordinates of the simulated B/Lee/40 model are available in ProteinData Bank with the accession code 1TX1. The pdb file was converted tothe MMDB (Molecular Modeling Data Base) format by using VAST (VectorAlignment Search Tool) search. The model of 1TX1 structure then wasviewed by Cn3D and the structure was saved in MMDB format. Based on thesame principal used to design influenza A HA1-1 and HA1-2 constructs,the domain boundaries of B/Lee/40 HA-1 and HA 1-2 were pinpointed byexamining the model structure. B/Lee/40 HA1-1 (SEQ ID NO: 38) includesthe epitope-concentrated globular top, the membrane distal β-sheet andan additional β-sandwich located underneath the membrane distal β-sheet.The amino terminus of HA1-1 starts from residue 48 and ends at residue340 (TTTPTK (SEQ ID NO: 279) . . . - . . . CPIWVK (SEQ ID NO: 280)) ofSEQ ID NO: 36). The B/Lee/40 HA1-2 (SEQ ID NO: 39) was designed byremoving the β-sandwich from HA1-1. HA1-2 starts from residue 60 andends at residue 299 (KGTQTR (SEQ ID NO: 281) . . . - . . . SKVIKG (SEQID NO: 282)) of SEQ ID NO: 36. In order to confirm the boundaryselection, other independent methods were also employed. The firstmethod used was the primary sequence alignment. The sequence ofA/Aichi/2/68 (SEQ ID NO: 35) was aligned with B/Lee/40 (SEQ ID NO: 36)sequence. The boundaries of A/Aichi/2/68 HA1-1 (SEQ ID NO: 40) (residues60-326 QSSSTG (SEQ ID NO: 283) . . . - . . . CPKYVK (SEQ ID NO: 284))and HA1-2 (SEQ ID NO: 41) (residues 72-287; HRILDG (SEQ ID NO: 285) . .. - . . . SIMRSD (SEQ ID NO: 286)) were aligned closely to those ofB/Lee/40, supporting the boundary selections using simulated model. Oneresidue adjustment was made to avoid exposing hydrophobic residues andeither end of the construct. Thus, the 3-dimensional structureprediction matches very well with primary sequence alignment.

The second method used was the secondary structure prediction. Thedomain boundary is usually located in the loop or turn region withoutinvading much of the secondary structure elements such as the α-helixand the β-sheet, especially the center of the α-helix bundle or theβ-sheet. This criterion was used to double check if the boundaryselection made form simulated 3-D structure is in agreement withindependent secondary structure prediction. The program PHD(http://ca.expasy.org/tools→Proteomics and sequence analysistools→Secondary and tertiary structure tools→PredictProtein) was used toperform the secondary structure prediction. Other than two of the HA1-2carboxy-terminal residues overlapping with a short α-helix (5 aminoacids), all other boundary residues fall in the loop regions, indicatinga reasonable boundary selection. PHD results is listed as the following,where AA is the amino acid sequence; OBS_sec is the observed secondarystructure: H=helix, E=extended sheet, blank=other (loop); PROF_sec: PROFpredicted secondary structure: H=helix, E=extended (sheet), blank=other(loop), PROF=PROF: Profile network prediction HeiDelberg; Rel_sec:reliability index for PROFsec prediction (0=low to 9=high)

For the brief presentation strong predictions marked by ‘*’; SUB_sec:subset of the PROFsec prediction, for all residues with an expectedaverage accuracy >82% NOTE: for this subset the following symbols areused: L: is loop (for which above ‘ ’ is used) .: means that noprediction is made for this residue, as the reliability is: Rel<5;O_(—)3_acc: observed relative solvent accessibility (ace) in 3 states:b=0-9%, i=9-36%, e=36-100%; P_(—)3_ace: PROF predicted relative solventaccessibility (ace) in 3 states: b=0-9%, i=9-36%, e=36-100%; Rel_acc:reliability index for PROFacc prediction (0=low to 9=high).

For the brief presentation strong predictions marked by ‘*’; SUB_acc:subset of the PROFacc prediction, for all residues with an expectedaverage correlation >0.69 (tables in header).

NOTE: for this subset the following symbols are used:

-   -   I: is intermediate (for which above ‘ ’ is used)    -   .: means that no prediction is made for this residue, as the        reliability is: Rel<4.

....,....1....,....2....,....3....,....4....,....5 AAMKAIIVLLMVVTSNADRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTT OBS_sec PROF_secEEEEEEEEEE     EEEEEEEE     EEEEEE    EEEEEEEEEE Rel_sec90456542111257762677454037753444441276147765653203 SUB_secL..EEE......LLLL.EEE.E...LLL........LL..EEEEEE.... O_3_accbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbb P_3_accee bbbbbbbbbeeb  bbbbbbb eeee b bb eeebeb  b  bbe Rel_acc33169997521111032778921201222233322135234021321012 SUB_acc...bbbbbb........bbbb................e..b.............,....6....,....7....,....8....,....9....,....10 AAPTKSHFANLKGTQTRGKLCPNCFNCTDLDVALGRPKCMGNTPSAKVSILH OBS_sec PROF_secEEEE                  EEEEEE              EEEE Rel_sec67752200245344675222033211232342043234337875203787 SUB_secLLLL......L...LLL.......................LLLL...EEE O_3_accbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbb P_3_acceee  bbe eeeeeee    eb  bbbbbbbbb b b ee eeeeb bbb Rel_acc23201011020002300210201031223343310100210221541265 SUB_acc..............................b.............eb..bb....,....11.1.,....12.1.,....13.1.,....14.1.,....15.1 AAEVKPATSGCFPIMHDRTKIRQLPNLLRGYENIRLSTSNVINTETAPGGPY OBS_sec PROF_secEE             HHHHHHHHHHH     EEEEE  EE Rel_sec50566441224778858887664221002304663021003444556531 SUB_secE.LLL......LLLLHHHHHHH..........EE..........LLLL.. O_3_accbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbb P_3_accb  eeee bbbbb   e b ebbebbee eeb         eeeeee Rel_acc12210110221001337264350314531332301200011120132011 SUB_acc................e.bi.b...be...........................,....16.1.,....17.1.,....18.1.,....19.1.,....20.1 AAKVGTSGSCPNVANGNGFFNTMAWVIPKDNNKTAINPVTVEVPYICSEGED OBS_sec PROF_secHHHHHHHHHHH            EEEEEE Rel_sec24431001255662036787888752378864236641012244157874 SUB_sec.........LLLL...HHHHHHHHH..LLLL...LL.........LLLL. O_3_accbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbb P_3_accbbbbbbee ee bbbbbbb  bbeeeee    ee ebeb b b eee Rel_acc11111012110301026314921522122321112112322203203212 SUB_acc................b..bb..b..............................,....21.1.,....22.1.,....23.1.,....24.1.,....25.1 AAQITVWGFHSDDKTQMERLYGDSNPQKFTSSANGVTTHYVSQIGGFPNQTE OBS_sec PROF_secEEEEEEEE     HHHHHH      EEEEE  EEEEEEEE Rel_sec47897532588742544320368853775200122343201275331035 SUB_sec.EEEEE..LLLL..H......LLLL.EEE.............LL.....L O_3_accbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbb P_3_accbbbbbbbbb eeee    b eeeeeeb b b e e e   ebee b   e Rel_acc19696993033223122210203213224011122211311001202001 SUB_acc.bbbbbb.....................b.........................,....26.1.,....27.1.,....28.1.,....29.1.,....30.1 AADEGLKQSGRIVVDYMVQKPGKTGTIVYQRGILLPQKVWCASGRSKVIKGS OBS_sec PROF_secEEEEEEEEE    EEEEEE   EEE  EEEEE    EEEE Rel_sec56754320377888983577247886434023022235520330245401 SUB_secLLLL.....EEEEEEE.LLL..EEEE...........EE.......E... O_3_accbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbb P_3_acceee  bbbbbbb bbbb ee  b b   bbbbbb  bb b eeb bbe e Rel_acc17211105270744553301222473010244520230221020211103 SUB_acc.e.....b.b.bibbb.......ib.....bbb.....................,....31.1.,....32.1.,....33.1.,....34.1.,....35.1 AALPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGTK OBS_sec PROF_secHHH    HHHHH HHHHH Rel_sec32233333000113432246764334340342334133220003551573 SUB_sec...................LLL......................HH.LL. O_3_accbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbb P_3_acce     e    e bb   ee  b      bbbebbe be bb bbbbb Rel_acc10010011112110110111101210102102111021712452461321 SUB_acc......................................b..bb.bb........,....36.1.,....37.1.,....38.1.,....39.1.,....40.1 AAYRPPAKLLKERGFFGAIAGFLEGGWEGMIAGWHGYTSHGAHGVAVAADLK OBS_sec PROF_secEEEEHHHH                     EEEEE  H Rel_sec56853211324203110113404531100120021023564325530623 SUB_secLLLL...................L..............LL...EE..L.. O_3_accbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbb P_3_accb  eee be  bbbbbbbbbbbbbb bbbbbbbbbbbbbb bbbbbbb e Rel_acc00011411202355999999623520456353043010011315264032 SUB_acc.....e......bbbbbbbbb..b..bbb.b..b.........b.bb.......,....41.1.,....42.1.,....43.1.,....44.1.,....45.1 AASTQEAINKITKNLNYLSELEVKNLQRLSGAMNELHDEILELDEKVDDLRA OBS_sec PROF_secHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH HHH Rel_sec47888989873212477733101234446677898888878756210111 SUB_sec.HHHHHHHHH.....HHH..........HHHHHHHHHHHHHHHH...... O_3_accbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbb P_3_accbbbebbeebbe b  bbee beebeeb ebbeebbe beeb eebee Rel_acc28049825725342322312063124534063444467553254223101 SUB_acc.b.ebb.eb.e.b........e...eb.e.b.ebbeibee..ee..........,....46.1.,....47.1.,....48.1.,....49.1.,....50.1 AADTISSQIELAVLLSNEGIINSEDEHLLALERKLKKMLGPSAVEIGNGCFE OBS_sec PROF_secHHHHHHHHHHHHHH         HHHHHHHHHHHHHHHHHHHH    EEE Rel_sec03645477777753464114550578879888887664424540663252 SUB_sec..H.H.HHHHHHH..L....LL.HHHHHHHHHHHHHH....H..LL..E. O_3_accbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbb P_3_accbbbb bb bbbbb  eebb  e e b eb e b e b e bee eebbbb Rel_acc04450592968651122001021323439443944173428232012340 SUB_acc.bbb.bb.bbbbb.............i.bie.bie.b.e.b.......b.....,....51.1.,....52.1.,....53.1.,....54.1.,....55.1 AATKHKCNQTCLDRIAAGTFNAGDFSLPTFDSLNITAASLNDDGLDNHTILL (SEQ ID NO: 36)OBS_sec PROF_sec EEEE  HHHHHHHH          HHHH  EEEEEEEEE      EEEEERel_sec 22003402344311355466523223103101002476217776303665 SUB_sec...............LL.LLL...............EE..LLLL...EEE O_3_accbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbb P_3_accb beb e bbbbb bbbb  ee  eebbe beb bbeb eeeeeeb bbb Rel_acc42120312620271012211330320211224131235303222133868 SUB_accb.......b...b..................e.....b.........bbb....,....56.1.,....57.1.,....58.1. (SEQ ID NO: 36) AAYYSTAASSLAVTLMIAIFIVYMVSRDNVSCSICL OBS_sec PROF_secEEEHHHHHHHHHHHHHHHHHHH     EEEEEEE Rel_sec4310021478888888776321006740346860 SUB_sec........HHHHHHHHHHH.....LL....EEE. O_3_accbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbb P_3_accbbbbbbbbbbbbbbbbbbbbbbbee eb b bbb Rel_acc6362366679999996998517101010152400 SUB_accb.b..bbbbbbbbbbbbbbb.b.......b.b..

The third tool that used is the hydrophobicity analysis. In general, thestructural core of a protein tends to be a hydrophobic cluster flankedby stretch of hydrophilic residues. Therefore the predicted boundariesshould not locate in the center of hydrophobic core, rather shouldreside in the flanking hydrophilic regions. Hydrophobicity of B/Lee/40HA sequence (SEQ ID NO: 36) was plotted using ProtScale(http://ca.expasy.org/tools→Primary structure analysis→ProtScale). Thehydrophobicity analysis of B/Lee/40 sequence confirmed that the selectedboundaries were in the hydrophilic regions of the protein (FIG. 2). Thehydrophobicity strength used in the plot is listed in the followingtable:

SEQUENCE LENGTH: 584 Using the scale Hphob./Kyte & Doolittle, theindividual values for the 20 amino acids are: Ala: 1.800 Arg: −4.500Asn: −3.500 Asp: −3.500 Cys: 2.500 Gln: −3.500 Glu: −3.500 Gly: −0.400His: −3.200 Ile: 4.500 Leu: 3.800 Lys: −3.900 Met: 1.900 Phe: 2.800 Pro:−1.600 Ser: −0.800 Thr: −0.700 Trp: −0.900 Tyr: −1.300 Val: 4.200 Asx:−3.500 Glx: −3.500 Xaa: −0.490

In order to identify the HA 1-1 and HA1-2 domain boundaries of otherinfluenza B molecules, such as B/Malaysia/2506/2004 (SEQ ID NO: 42),B/Ohio/1/2005 (SEQ ID NO: 43), B/Victoria/2/87 lineage (SEQ ID NO: 787)and B/Shanghai/361/2002 (SEQ ID NO: 44), and B/Yamagata/16/88 lineage(SEQ ID NO: 213), each HA sequence was aligned with B/Lee/40 HA sequence(SEQ ID NO: 36). The results indicated that influenza B HA proteins aremuch more conserved compared to influenza A HA proteins, especially inthe domain boundary sequences. Thus, the boundaries identified inB/Lee/40 were used to direct boundary selections of other influenza Bstrains. The resulting domain boundaries for each strain are summarizedbelow.

B/Malaysia/2506/2004: HA1-1 44-337 (SEQ ID NO: 45) (TTTPTK (SEQ ID NO:287)-CPIWVK (SEQ ID NO: 288)) HA1-2 56-296 (SEQ ID NO: 46) (KGTETR (SEQID NO: 289)-SKVIKG (SEQ ID NO: 290))

B/Ohio/1/2005: HA1-1 33-326 (SEQ ID NO: 47) (TTTPTK (SEQ ID NO:291)CPIWVK (SEQ ID NO: 292)) HA1-2 45-285 (SEQ ID NO: 48) (KGTKTR (SEQID NO: 293)SKVIKG (SEQ ID NO: 294))

B/Shanghai/361/2002: HA1-1 33-325 (SEQ ID NO: 49) (TTTPIK (SEQ ID NO:295)CPIWVK (SEQ ID NO: 296)) HA1-2 45-284 (SEQ ID NO: 50) (KGTRTR (SEQID NO: 297)SKVIKG (SEQ ID NO: 298))

BMal ----IVLLMVVTSNADRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTT 50 BOhi---------------DRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTT BLeeMKAIIVLLMVVTSNADRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTT BSha---------------DRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTT               *********************************** BMalPTKSHFANLKGTETRGKLCPKCLNCTDLDVALGRPKCTGNIPSARVSILH 100 BOhiPTKSHFANLKGTKTRGKLCPKCLNCTDLDVALGRPKCTGNIPSAEVSILH BLeePTKSHFANLKGTQTRGKLCPNCFNCTDLDVALGRPKCMGNTPSAKVSILH BShaPIKSHFANLKGTRTRGKLCPDCLNCTDLDVALGRPMCVGTTPSAKASILH* **********.*******.*:************ * *. ***..**** BMalEVRPVTSGCFPIMHDRTKIRQLPNLLRGYEHIRLSTHNVINAENAPGGSY 150 BOhiEVRPVTSGCFPIMHDRTKIRQLPNLLRGYEHIRLSTHNVINAEKAPGGPY BLeeEVKPATSGCFPIMHDRTKIRQLPNLLRGYENIRLSTSNVINTETAPGGPY BShaEVRPVTSGCFPIMHDRTKIRQLPNLLRGYENIRLSTQNVIDAEKALGGPY**:*.*************************:***** ***::*.* **.* BMalKIGTSGSCPNVTNGNGFFATMAWAVPKNDNNKTATNSLTIEVPYICTEGE 200 BOhiKIGTSGSCPNVTNGNGFFATMAWAVPKNDNNKTATNSLTIEVPYICTEGE BLeeKVGTSGSCPNVANGNGFFNTMAWVIPK-DNNKTAINPVTVEVPYICSEGE BShaRLGTSGSCPNATSKSGFFATMAWAVPK-DNNKNATNPLTVEVPYICTEGE::********.:. .*** ****.:** ****.* *.:*:******:*** BMalDQITVWGFHSDNEAQMAKLYGDSKPQKFTSSANGVTTHYVSQIGGFPNQT 250 BOhiDQITIWGFHSDSETQMAKLYGDSKPQKFTSSANGVTTHYVSQIGGFPNQT BLeeDQITVWGFHSDDKTQMERLYGDSNPQKFTSSANGVTTHYVSQIGGFPNQT BShaDQITVWGFHSDDKTQMKNLYGDSNPQKFTSSANGVTTHYVSQIGGFPDQT****:******.::** .*****:***********************:** BMalEDGGLPQSGRIVVDYMVQKSGKTGTITYQRGILLPQKVWCASGRSKVIKG 300 BOhiEDGGLPQSGRIVVDYMVQKSGKTGTITYQRGILLPQKVWCASGRSKVIKG BLeeEDEGLKQSGRIVVDYMVQKPGKTGTIVYQRGILLPQKVWCASGRSKVIKG BShaEDGGLPQSGRIVVDYMVQKPGKTGTIVYQRGVLLPQKVWCASGRSKVIKG** ** *************.******.****:****************** BMalSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGT 350 BOhiSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGT BLeeSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGT BShaSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGHCPIWVKTPLKLANGT**********************************:*************** BMalKYRPPAKLLKER-------------------------------------- 400 (SEQ ID NO: 42)BOhi KYRPPAKLLKERGF------------------------------------ (SEQ ID NO: 43)BLee KYRPPAKLLKERGFFGAIAGFLEGGWEGMIAGWHGYTSHGAHGVAVAADL (SEQ ID NO: 36)BSha KYRP---------------------------------------------- (SEQ ID NO: 44)****

B/Lee/40 HA1-1 Construct Design (SEQ ID NO: 38).

For this construct, five (5) anticipated conserved disulfide bonds werepreserved by making the amino terminal truncation before the Cysteine atposition 69 and the carboxyl-terminal truncation after the Cysteine atposition 335 of SEQ ID NO: 36. The likely disulfide pairings are as thefollowing: C69 and C72, C75 and C87, C109 and C158, C195 and C289, C309and C335 of SEQ ID NO: 36. Using the simulated structures and the MMDBto check secondary structures the amino terminal truncation was made atthe Theonine at position 48, right after the β-strand kink introduced byProline 46 followed by Leucine 47 of SEQ ID NO: 36, such that thepreceding β-strand [TGVI, SEQ ID NO: 299, residues 42-45 of SEQ ID NO:36] was totally eliminated while the entire short β-strand formed byresidues TTTP (SEQ ID NO: 300) [residues 48-51 of SEQ ID NO: 36] waspreserved. The carboxyl-terminal truncation was made at the end ofβ-strand PIWV (SEQ ID NO: 301) [residues 336-340 of SEQ ID NO: 36]following the Cysteine at position 335 such that the carboxyl-terminaltail which interacts with HA2 was completely eliminated. Six β-strandsincluding the C-terminal β-strand form a stable β-sandwich defined byβ-strands PIWV (SEQ ID NO: 301) [residue 336-340 of SEQ ID NO: 36],PYYTG (SEQ ID NO: 302) [residues 322 to 326 of SEQ ID NO: 36] and TTTP(SEQ ID NO: 300) [residues 48-51 of SEQ ID NO: 36] (lower β-pleatedsheet), and the β-strands DCLHE (SEQ ID NO: 303) [residues 308-312 ofSEQ ID NO: 38], YGGLN (SEQ ID NO: 304) [residue 314-318 of SEQ ID NO:36] and KAIGN (SEQ ID NO: 305) [residues 330-334 of SEQ ID NO: 36] (topβ-pleated sheet). This set of β-pleated sheet includes both theamino-terminus and carboxyl-terminus within one stable secondarystructure element. Such truncations were made to believe resulting acompact domain structure.

B/Lee/40 HA1-2 Construct Design (SEQ ID NO: 39).

For this construct four (4) anticipated conserved disulfide bonds werepreserved by making the amino-terminal truncation before the Cysteine atposition 69 and the carboxyl-terminal truncation after the Cysteine atposition 289 of SEQ ID NO: 36. Using the simulated structure and theMMDB to check secondary structures the amino-terminal truncation wasmade in the loops that connect two distinct sets of β-pleated sheets.The amino-terminal truncation was made at the Lysine at position 60,such that the β-strand comprising residues 62-66 of SEQ ID NO: 36[TQTRG, SEQ ID NO: 306] of the membrane-distal set of β-strands remainedwholly intact, while the membrane-proximal β-strand [TTTP, SEQ ID NO:300, residues 48-51 of SEQ ID NO: 36] that forms part of HA1-1 wascompletely eliminated. The carboxyl-terminal truncation was made at theSerine 300 of SEQ ID NO: 36. Truncation at this position preservedsecondary structure such that the membrane-distal β-strand [KVIKG, SEQID NO: 307, residues 295-299 of SEQ ID NO: 36] remained intact while themembrane-proximal β-strand [DCLHE, SEQ ID NO: 308, residues 308-312 ofSEQ ID NO: 36] was eliminated. The membrane-distal β-pleated sheetscomprising β-strands TQTRG (SEQ ID NO: 306) [residues 62-66 of SEQ IDNO: 36], SILHEV (SEQ ID NO: 309) [residues 97-102 of SEQ ID NO: 36], andKVIKG (SEQ ID NO: 307) [residues 295-299 of SEQ ID NO: 36] serve as thestabilizing secondary structure element that is believed to conclude acompact domain structure.

B/Malaysia/2506/2004, B/Ohio/1/2005 and B/Shanghai/361/2002 follow thesame reference structure as described above.

Example 3 Cloning and Expression of Recombinant Flagellin-HemagglutininFusion Proteins in E. coli Materials and Methods

Cloning of HA Influenza A Subunits.

Subunits of the HA globular head from several strains of influenza Awere cloned and expressed alone or as fusions with flagellin. We alsoexpressed in fusion with the HA globular head domain, two proteinswidely used as carrier proteins in conjugated vaccines. CRM-197 is amutated diphtheria toxin (DTx) from Corynebacterium diphtheriae and LTB,is E. coli heat labile toxin B subunit.

These constructs were generated in one of four different methodologies:

Method #1:

In this protocol, a fusion gene comprising flagellin (STF2) (SEQ ID NO:212) and the HA subunit was codon-optimized for E. coli expression andobtained from a commercial vendor (DNA2.0 Inc., Menlo Park, Calif.) bychemical synthesis. The gene was excised with NdeI and BlpI enzymes, theinsert fragment was gel purified and ligated to pET24a (Novagen, SanDiego, Calif.) which had been digested with NdeI and BlpI and treatedwith bacterial alkaline phosphatase (BAP).

Method #2:

For facile cloning of genes in fusion with flagellin, a cassette plasmidcontaining a unique BlpI site at the 3′ end of the flagellin (STF2) gene(SEQ ID NO: 212) was generated. This was done by introducing a silentmutation at nucleotides 5′-GTGCTGAGCCTGTTACGT-3′ (SEQ ID NO: 310) [nt1501 to 1518 of SEQ ID NO: 212] of STF2 creating the unique BlpI site inthe plasmid cassette, pET24/STF2.blp (SEQ ID NO: 51). Synthetic genesfor each target antigen were codon-optimized for E. coli expression andobtained from a commercial vendor (DNA2.0 Inc., Menlo Park, Calif.). Thesynthetic genes were excised with BlpI enzyme and ligated by compatibleends to pET24/STF2.blp which had been treated with BlpI and BAP.

Method #3:

Using forward and reverse primers (Keck Foundation BRLK, YaleUniversity, New Haven, Conn.; Midland Certified Reagent Company,Midland, Tex.) as indicated for each construct, PCR amplification wasperformed using the DNA templates shown in each table. The PCR productwas subjected to BlpI digestion, gel-purified and ligated topET24/STF2.blp (SEQ ID NO: 51) vector previously prepared by BlpIdigestion and BAP treatment.

Method #4:

A plasmid cassette, pET24/STF2.5G (SEQ ID NO: 52), was generated byintroducing a flexible heptamer linker, Ser-Gly-Ser-Gly-Ser-Gly-Ser(S-G-S-G-S-G-S (SEQ ID NO: 311)) at the 3′ end of the STF2 gene. Aunique BamHI site was created in the linker to facilitate cloning of HAsubunit fragments in fusion with flagellin. Synthetic genescodon-optimized for expression in E. coli were obtained from acommercial vendor (DNA2.0 Inc., Menlo Park, Calif.), excised from theparental plasmids with BamHI and BlpI restriction endonucleases, andligated by compatible ends to the pET24/STF2 cassette that hadpreviously been digested with BamHI and BAP-treated.

In each case, the constructed plasmids were used to transform competentE. coli TOP10 cells and putative recombinants were identified by PCRscreening and restriction mapping analysis. The integrity of theconstructs was verified by DNA sequencing and they were used totransform the expression host, BLR3 (DE3) (Novagen, San Diego, Calif.;Cat #69053). Transformants were selected on plates containing kanamycin(50 μg/mL), tetracycline (5 μg/mL) and glucose (0.5%). Colonies werepicked and inoculated into 2 ml of LB medium supplemented with 25 μg/mlkanamycin, 12.5 μg/ml tetracycline and 0.5% glucose and grown overnight.Aliquots of these cultures were used to inoculate fresh cultures in thesame medium formulation, which were cultured until they reached anOD₆₀₀=0.6, at which time protein expression was induced by the additionof 1 mM IPTG and culturing for 3 hours at 37° C. The cells wereharvested and analyzed for protein expression.

SDS-PAGE and Western Blot:

Protein expression and identity were determined by gel electrophoresisand immunoblot analysis. Cells were harvested by centrifugation andlysed in Laemmli buffer. An aliquot of 10 μl of each lysate was dilutedin SDS-PAGE sample buffer with or without 100 mM DTT as a reductant. Thesamples were boiled for 5 minutes and loaded onto a 10% SDSpolyacrylamide gel and electrophoresed (SDS-PAGE). The gel was stainedwith Coomassie R-250 (Bio-Rad; Hercules, Calif.) to visualize proteinbands. For western blot, 0.5 μl/lane cell lysate was electrophoresed andelectrotransferred to a PVDF membrane and blocked with 5% (w/v) drymilk.

The membrane was then probed with either anti-flagellin antibody(Inotek; Beverly, Mass.) or influenza A PR/8/38 convalescent immunemouse serum. PR/8/34 immune serum was generated in BALB/c mice (JacksonLaboratory, Bar Harbor, Me.) that received an experimentally determinedsublethal challenge dose of 8×10¹ egg infectious dosages (EID) ofPR/8/34 influenza virus. Animals were then allowed to convalesce for >21days post-infection at which time immune serum was isolated andclarified. After probing with alkaline phosphatase-conjugated secondaryantibodies (Pierce; Rockland, Ill.), protein bands were visualized withan alkaline phosphatase chromogenic substrate (Promega, Madison, Wis.).Bacterial clones which yielded protein bands of the correct molecularweight and reactive with the appropriate antibodies were selected forproduction of protein for use in biological assays.

The constructs derived from the HA of A/Puerto Rico/8/34 strain (PR8)(SEQ ID NO: 1) and listed in Table 1 were made by the synthetic generoute as described in Method #1 and Method #4. Similarly, the constructsderived from the A/Viet Nam/1203/2004 strain (SEQ ID NO: 2) are shown inTable 2, the constructs derived from the HA of A/Indonesia/2005 strain(IND) (SEQ ID NO: 3) are described in Table 3, and those from A/NewCaledonia/12/99 strain (NC) (SEQ ID NO: 4) are described in Tables 4.“IND” as used herein, refers to “Indonesia.” Where appropriate, the DNAprimers and DNA templates used in the PCR amplification reaction arelisted in the same table.

TABLE 1 PR8 HA constructs for expression in E coli SEQ ID NO: ConstructMethod 53 STF2.HA1-1 #4 54 STF2.HA1-1.his #4 55 STF2.HA1-2 #1 56STF2.HA1-2mut #1 57 STF2.HA1-3 #1 58 STF2.HAl-3mut #1 59 HA1-1 #1 60HA1-1.his #1 61 HA1-2.his #1 62 CRM.HA1-2 #1 63 LTB.HA1-2 #1

TABLE 2 VN HA constructs for expression in E coli SEQ FOR REV DNA IDPrimer Primer Template NO: Construct Method SEQ ID NO: SEQ ID NO: SEQ IDNO: 64 STF2.HA1-1 #3 65 66 67 68 STF2.HA1-2 #3 69 70 67 71 STF2.HA1- #2N/A N/A 72 2mut

TABLE 3 IND HA constructs for expression in E coli SEQ FOR REV DNA IDPrimer Primer Template NO: Construct Method SEQ ID NO: SEQ ID NO: SEQ IDNO: 73 STF2.HA1-1 #3 74 66 75 76 STF2.HA1-2 #3 69 77 75 78 STF2.HA1- #2N/A N/A 79 2mut

TABLE 4 NC HA constructs for expression in E coli SEQ FOR REV DNA IDPrimer Primer Template NO: Construct Method SEQ ID NO: SEQ ID NO: SEQ IDNO: 80 STF2.HA1-1 #3 81 82 83 84 STF2.HA1-2 #3 85 86 83 87 STF2.HA1- #2N/A N/A 88 2mut

Results

Protein carriers have widespread application in human vaccines. Thecross-reactive material (CRM₁₉₇) of diphtheria toxin is considered to beadvantageous as a carrier molecule in the formulation of severalconjugate vaccines. Exemplary carriers include E. coli heat labileenterotoxin (LT) and its B subunit (LTB), Tetanus toxoid (TT) andcholera toxin (CT). Using CRM₁₉₇ and LTB as representatives of thisgroup of carrier proteins, we have generated constructs in which theglobular head of HA of (A/Puerto Rico/8/34 strain (PR8) (SEQ ID NO: 1)is fused to the 3′ end of either CRM197 gene or LTB gene as described inMethod #1 generating the constructs CRM.HA1-2 (SEQ ID NO: 62) andLTB.HA1-2(SEQ ID NO: 63). The constructs were verified by DNA sequencingand used to transform the expression host, BLR3 (DE3) (Novagen, SanDiego, Calif.; Cat #69053).

Transformants were selected on plates containing kanamycin (50 μg/mL),tetracycline (5 μg/mL) and glucose (0.5%). Several colonies were pickedfor an overnight culture which were was used to inoculate a fresh LBmedium supplemented with 25 μg/ml kanamycin, 12.5 μg/ml tetracycline and0.5% glucose. At an OD600=0.6 protein expression was induced with 1 mMIPTG for 3 h at 37° C. The cells were harvested and an aliquot of thelysate was analyzed on 10% SDS-PAGE by Coomassie blue staining and byimmunoblot using PR/8/34 convalescent sera. In the case of CRM.HA1-2construct several clones were picked and analyzed for expression bySDS-PAGE.

As assayed by Coomassie blue staining of the SDS-PAGE gel, all theclones displayed a band that migrated with an apparent MW 84 KDa andthat corresponds to the predicted MW. The absence of this band in thecontrol culture (without IPTG) indicates that it is specifically inducedby IPTG. This observation was further confirmed when the cell extractsof two clones #5 and #6 was fractionated in the presence or absence of areducing agent (5 mM DTT). While the recombinant protein whosedisulphide bonds have been disrupted by treatment with DTT is notrecognized by the cognate antibodies, the native recombinant protein is.Similarly, a clone expressing construct LTB.HA1-2 displays a bandcorresponding to the predicted molecular weight of 39.8 KDa, proteinwhen induced with IPTG. The identity of LTB-HA1-2 fusion protein isconfirmed by western blot analysis using mouse convalescent serum. Thisband is diminished in intensity when a reductant (β-mercaptoethanol) waspresent. This latter observation suggests that insufficient amount ofreductant was most likely employed in the experiment. Taken together thedata presented herein support the notion that the globular head of HAcan be successfully fused to carrier proteins to generate conformationalsensitive proteins.

Cloning of Recombinant Flagellin-Hemagglutinin Fusion Proteins in E.coli

Cloning of HA Influenza B Subunits.

Subunits of the HA globular head from several strains of influenza Bwere cloned and expressed as fusions with flagellin. These constructswere generated by a two-step PCR.

The HA subunit was codon-optimized for E. coli expression and obtainedfrom a commercial vendor (DNA2.0 Inc., Menlo Park, Calif.) by chemicalsynthesis. The HA1-1 or HA1-2 was PCR amplified using the synthesizedDNA as templates. The flagellin (STF2) sequence (SEQ ID NO: 212) wasderived from the plasmid pET24a-STF2.HA1-2. The STF2 DNA fragment wasPCR amplified, and the C-terminal of the PCR product has a 28-30 bpoverlap with the N-terminal sequence of the fusion HA subunit.

The STF2 and HA subunits were fused together by a 2^(nd) PCR. Usingrespective forward and reverse primers (Integrated DNA Technologies,Inc, Coralville, Iowa 52241) listed below, fusion protein DNA wasamplified from the DNA templates also shown below. The PCR product wassubsequently subjected to XbaI digestion, gel-purification and ligationto pET24a-STF2.HA1-2 that was previously digested with XbaI and SnaBI.

The constructs are listed below, where appropriate, the DNA primers andDNA templates used in the PCR amplification reaction are also listed.

FluB STF2.HA constructs for expression in E coli SEQ FOR REV DNA IDPrimer Primer Template NO: Construct SEQ ID NO: SEQ ID NO: SEQ ID NO:184 STF2.HA1-1 (MAL) 193, 195 194, 196 55, 183 186 STF2.HA1-2 (MAL) 193,198 197, 199 55, 185 188 STF2.HA1-2 (SH) 193, 201 200, 202 55, 187 190STF2.HA1-2 (Lee) 193, 204 203, 205 55, 189 192 STF2.HA1-2 (Ohio) 193,207 206, 208 55, 191

In each case, the constructed plasmids were used to transform competentE. coli DH5α cells and putative recombinants were identified byrestriction mapping. The integrity of the constructs was verified by DNAsequencing and was used to transform the expression host, BLR3 (DE3)(Novagen, San Diego, Calif.; Cat #69053). Transformants were selected onLB (Veggie peptone, EMD Bioscience, La Jolla, Calif.; Cat#71280; veggieyeast extract, EMD Bioscience, Cat#71279;) plates containing kanamycin(35 μg/mL). Colonies were picked and inoculated into 2 ml of LB (Veggie)medium supplemented with 35 μg/ml kanamycin and grown overnight.Aliquots of these cultures were used to inoculate fresh cultures in thesame medium formulation, which were cultured until OD₆₀₀=0.6 wasreached. The protein expression was induced by the addition of 1 mMIPTG. The cells were harvested after 2 hours culturing at 37° C. andanalyzed for protein expression.

SDS-PAGE and Western Blot:

Protein expression and identity were determined by gel electrophoresisand immunoblot analysis. Cells were harvested by centrifugation andlysed in Tris/NaCl (50 mM Tris, 200 mM NaCl, pH 8.0) buffer. Twoaliquots of each lysate was diluted in SDS-PAGE sample buffer and boiledfor 5 minutes. One aliquot sample was reduced by DTT (200 mM finalconcentration, EMD Bioscience, La Jolla, Calif. 92039; Cat#233155). Thesamples (1.5 μl/lane cell lysate) were loaded onto a 4-12% SDSpolyacrylamide gel and electrophoresed (SDS-PAGE). The gel was stainedwith Coomassie R-250 (Bio-Rad; Hercules, Calif.) to visualize proteinbands.

For western blot, the duplicated gel was electrophoresed andelectrotransferred to a PVDF membrane and blocked with 5% (w/v) drymilk. The membrane was then probed with either anti-flagellin antibody(6H11, Inotek, Beverly, Mass.; Lot#1030B5) or ferret antiserum raisedagainst B/Malaysia (2506/2004, CDC#2005741987). After probing withHorseradish Peroxidase (HRP)-conjugated secondary antibodies (1:10,000)(Goat anti-Mouse is from Jackson ImmunoResearch Laboratories; WestGrove, Pa. Goat anti-Ferret is from Bethyl Laboratories; Montgomery,Tex.), protein bands were visualized after with addition of TMB, an HRPchromogenic substrate (Pierce; Rockland, Ill.; Cat#34018). Theapproximate molecular weight of STF2.HA1-1(MAL) is 84 kDa; and theapproximate molecular weight of STF2.HA1-2 (MAL and SH) is ˜78 kDa. Twomicrograms of STF2.4×M2e fusion protein was loaded as a positive controlfor the anti-flagellin antibody and a negative control for the anti-HAsera. Virus lysate (B/Malaysia/2506/04, CDC#2005756250) was included inthe experiment as a positive control for the anti-HA sera and a negativecontrol for the anti-flagellin antibody. Commassie blue staining of thegel showed low induction of STF2.HA1-1(MAL) protein; but much strongerbands for STF2.HA1-2(MAL) and STF2.HA1-2(SH) proteins. Western blotanalysis using anti-flagellin monoclonal antibodies (1:4000) exhibited astrong positive band around 84 kDa for lanes loaded withSTF2.HA1-1(MAL), and around 75 kDa in lanes loaded with STF2.HA1-2(MAL)or STF2.HA1-2(SH). The induction pattern of each fusion protein on theWestern is very similar to the result for the commassie blue stainedgel. The level of the non-reduced and the reduced protein recognized bythe anti-flagellin was comparable. The positive control foranti-flagellin (STF2.4×M2e) showed s single strong band, and thenegative control exhibited no signal.

Western blot analysis using anti-HA polyclonal anti-sera (1:1000)exhibited a similar pattern as the blot with anti-flagellin, except thatthe reduced STF2.HA1-2 protein had a diminished signal compared to thatof the non-reduced sample, indicating that a substantial portion of theanti-HA polyclonal antibodies in the antiserum recognize conformationalepitopes formed by correct disulfide-bond formation while a smallproportion of the antibodies in the anti-sera recognize linear epitopesthat are not reliant on disulfide bound formation. Similar were observedusing a capture ELISA assay. It is worth noting that anti-serum againstB/Malaysia/2506/2004 (Victoria lineage) also recognizesB/Shanghai/361/2002, which belongs to Yamagata lineage. This resultindicates that these two lineages not only share substantial primarysequence similarity, but also share tertiary structure.

The western blot and ELISA results confirm the expression of functionalInfluenza B HA1-1 and HA1-2 fusion proteins in E. coli.

Example 4 Purification and Biochemical Characterization of RecombinantFlagellin-Hemagglutinin Fusion Proteins Materials and Methods

Bacterial Growth and Cell Lysis:

HA constructs were expressed in the E. coli host strain BLR (DE3). E.coli cells carrying a plasmed encoding STF2.HA1-1.his(PR8) (SEQ ID NO:54) were cultured and harvested as described above. Individual strainswere retrieved from glycerol stocks and grown in shake flasks to a finalvolume of 12 L. Cells were grown in LB medium containing 50 μg/mlkanamycin/12.5 μg/ml tetracycline/0.5% dextrose to OD₆₀₀=0.6 and inducedwith 1 mM IPTG for 3 h at 37° C. The cells were harvested bycentrifugation (7000 rpm×7 minutes in a Sorvall RC5C centrifuge) andresuspended in 1×PBS, 1% glycerol, 1 μg/ml DNAseI, 1 mM PMSF, proteaseinhibitor cocktail and 1 mg/ml lysozyme. The cells were then lysed bytwo passes through a microfluidizer at 15,000 psi. The lysate wascentrifuged at 45,000 g for one hour in a Beckman Optima Lultracentrifuge (Beckman Coulter; Fullerton, Calif.) to separate thesoluble and insoluble fractions

Purification and Refolding of STF2.HA1-1.his(PR8) (SEQ ID NO: 89)Protein from E. coli.

Following lysis and centrifugation, the insoluble (pellet) fraction wasresuspended in 100 ml 1×TBS, pH 8.0+1 mM β-mercaptoethanol and solidurea was added to a final concentration of 6.2 M. After resuspensionwith a Dounce glass-ball homogenizer the mixture was centrifuged asdescribed above. The resulting supernatant was loaded onto a chelatingsepharose column (GE/Amersham Biosciences; Piscataway, N.J.) chargedwith NiSO₄ and equilibrated in Buffer A [20 mM Tris, pH 8.0/8 M urea/0.5M NaCl].

After washing with 1 L buffer B [Buffer A+1% (w/v) TX-100] the columnwas eluted in a 5-column volume linear gradient from 100% Buffer A to100% Buffer C [Buffer A+0.5 M imidazole]. Peak fractions were pooled,concentrated 3.5 fold on an Amicon 15 spin concentrator (Millipore;Billerica, Mass.) and dialyzed against 3×2 L of 8 M urea/20 mM Tris, pH8.0/2 mM EDTA. Following dialysis, STF2.HA1-1.his(PR8) (SEQ ID NO: 89)was refolded by rapid dilution to a final concentration of 0.1 mg/mlprotein in Refolding Buffer [0.1 M Tris, pH 8.0/0.1 M NaCl/1% (w/v)glycerol/5 mM reduced glutathione/1 mM oxidized glutathione] andincubated overnight at room temperature.

The refolded protein was then captured on a 5 ml chelating sepharoseHiTrap column (GE/Amersham Biosciences; Piscataway, N.J.) charged withNiSO₄ and equilibrated in Buffer D [20 mM Tris, pH 8.0/0.5 M NaCl/1 mMreduced glutathione/0.2 mM oxidized glutathione] and eluted in 100%Buffer E [Buffer D+0.5 M imidazole]. Peak eluate fractions were pooled,concentrated using an Amicon 15 spin concentrator (Millipore; Billerica,Mass.) and fractionated on a Superdex 200 size-exclusion column(GE/Amersham; Piscataway, N.J.) equilibrated in 1×TBS, pH 8.0+0.5% (w/v)Na deoxycholate. 5200 peak fractions were pooled, dialyzed against 3×2 Lof 1×TBS, pH 8.0 (without deoxycholate), vialed and stored at −80° C.

Purification of STF2.HA1-2(PR8) (SEQ ID NO: 90) and STF2.HA1-2mut(PR8)(SEQ ID NO: 91):

E. coli cells carrying a plasmid encoding STF2.HA1-2.his (PR8) (SEQ IDNO: 55) were cultured and harvested as described above. Following lysisand centrifugation, the insoluble (pellet) fraction containingSTF2.HA1-2(PR8) was resuspended and homogenized in 50 mM Tris, pH8.0+0.5% (w/v) Triton X-100 using a Dounce homogenizer. The homogenatewas then centrifuged (19,000 rpm×10 min). The resulting pellet fractionwas then re-homogenized in 50 mM Tris, pH 8.0+0.5% (w/v) TritonX-100+1.0 M NaCl and centrifuged. The inclusion body fraction (pellet)was then dissolved in buffer A [50 mM Na Acetate, pH 4.0+8.0 M urea] andcentrifuged (19,000 rpm×10 min.) to remove insoluble debris.

The resulting supernatant was fractionated on a Source S column(GE/Amersham Biosciences; Piscataway, N.J.) equilibrated in Buffer A andeluted in a 5 column-volume linear gradient with Buffer B (50 mM NaAcetate, pH 4.0, 1.0 M NaCl). The protein was then refolded by rapiddilution into Buffer C (100 mM Tris-HCl, pH 8.0). The refolded proteinwas then fractionated on a Q sepharose HP column (GE/AmershamBiosciences; Piscataway, N.J.) equilibrated in Buffer C and eluted in a0% to 60% linear gradient with buffer D (Buffer C+1 M NaCl). The Q HPeluate was then fractionated on a Superdex 200 size exclusionchromatography (SEC) column (GE/Amersham Biosciences; Piscataway, N.J.)equilibrated with 1× Tris-buffered saline, pH 8.0, to separate monomericfrom aggregated protein. Monomeric fractions were then pooled, aliquotedand stored at −80° C.

Purification of STF2.HA1-3(PR8) (SEQ ID NO: 92):

E. coli cells carrying a plasmed encoding STF2.HA1-3 (PR8) (SEQ ID NO:57) were cultured and harvested as described above. Following lysis andcentrifugation the soluble (supernatant) fraction containingSTF2.HA1-3(PR8) was adjusted to 6.2 M urea, 100 mM DTT and incubated atroom temperature overnight to completely reduce and denature theprotein. The protein was then diluted five-fold in Buffer A (50 mMcitric acid, pH 3.5, 8 M urea, 1 mM β-mercaptoethanol, 1 mM EDTA) andloaded onto an SP sepharose FF column (GE/Amersham Biosciences;Piscataway, N.J.) equilibrated with Buffer A. To remove endotoxin, thecolumn was washed with 10 column volumes of Buffer B (Buffer A+1% (w/v)Triton X-100) followed by 10 column volumes Buffer C (50 mM citric acid,pH 3.5, 1 mM β-mercaptoethanol, 1 mM EDTA, 70% (v/v) isopropanol).

The bound protein was then eluted in a five column-volume lineargradient of Buffer A:Buffer D (Buffer A+1M NaCl). The eluted protein wasthen dialyzed to Buffer E (20 mM Tris, pH 8.5, 8 M urea, 1 mMβ-mercaptoethanol, 1 mM EDTA) and passed through a Source Q column(GE/Amersham Biosciences; Piscataway, N.J.). The STF2.HA1-3(PR8) (SEQ IDNO: 92) protein was collected in the flow-thru fraction. The protein wasconcentrated using Amicon spin-concentrators (Millipore; Billerica,Mass.) and dialyzed to Buffer F (20 mM Tris, pH 8.0, 8 M urea, 1 mMEDTA) to remove reductant. The denatured polypeptide was then refoldedby rapid dilution to a final concentration of 0.1 mg/ml in RefoldingBuffer (0.1 M Tris, pH 8.0, 0.1 M NaCl, 1% (w/v) glycerol, 5 mM reducedglutathione, 1 mM oxidized glutathione) and incubated overnight at roomtemperature. The refolded protein was then fractionated on a Superdex200 size exclusion column equilibrated in 1× Tris-buffered saline (TBS),pH 8.0, plus 0.25% (w/v) sodium deoxycholate to separate monomericSTF2.HA1-3(PR8) from aggregated protein. Monomeric peak fractions werepooled, dialyzed to 1×TBS, pH 8.0, to remove deoxycholate, aliquoted andstored at −80° C.

Purification of HA1-2His(PR8) (SEQ ID NO: 93):

E. coli cells carrying a plasmed encoding STF2.HA1-2.his (PR8) (SEQ IDNO: 61) were cultured and harvested as described above. Following celllysis the insoluble fraction was homogenized and washed once with BufferA (50 mM Tris, pH 8.0), followed by sequential washing with BufferA+0.5% (w/v) Triton X-100 and Buffer A+0.5% (w/v) Triton X-100+1M NaCl.The insoluble material was then washed twice more with Buffer A alone.The washed inclusion bodies were then dissolved in Buffer B (1×PBS, pH7.4, 8 M urea) and applied to a Ni-NTA (Sigma-Aldrich; St. Louis, Mo.)column charged with NiSO₄ and eluted in a linear gradient of Buffer C(50 mM Tris, pH 8.0, 8 M urea, 0.3 M NaCl, 0.5 M imidazole). In order toreduce endotoxin, the eluted protein was dialyzed overnight against1×PBS, pH 7.4, 8 M urea, and reapplied to the Ni-NTA column.

The column was then washed with Buffer D (10 mM Tris, pH 8.8, 8 M urea,60% (v/v) isopropanol) and eluted as described above. Ni-NTA purifiedprotein was then refolded by 1:10 (v/v) dilution into Refolding Buffer(50 mM Tris, pH 8.8). The refolded HA1-2 His(PR8) (SEQ ID NO: 93)protein was then concentrated using an Amicon ultrafiltration unit(Millipore; Billerica, Mass.) and applied to a Superdex 200 sizeexclusion column (GE/Amersham; Piscataway, N.J.) equilibrated in 1×TBS,pH 8.0. Monomeric peak fractions were pooled, aliqoted and stored at−80° C.

SDS-PAGE and Western Blot Analysis:

Protein identity of all HA constructs was determined, and purityestimated, by SDS-PAGE. An aliquot of 5 μg of each sample was diluted inSDS-PAGE sample buffer with or without 100 mM DTT as a reductant. Thesamples were boiled for 5 minutes and loaded onto a 10% SDSpolyacrylamide gel (LifeGels; French's Forrest, New South Wales, AUS)and electrophoresed. The gel was stained with Coomassie R-250 (Bio-Rad;Hercules, Calif.) to visualize protein bands. For western blot, 0.5μg/lane total protein was electrophoresed as described above and thegels were then electro-transferred to a PVDF membrane and blocked with5% (w/v) dry milk before probing with anti-flagellin antibody (Inotek;Beverly, Mass.) or influenza A PR/8/34 Convalescent immune serum(described below under Protein Antigenicity ELISA). After probing withalkaline phosphatase-conjugated secondary antibodies (Pierce; Rockland,Ill.), protein bands were visualized with an alkaline phosphatasechromogenic substrate (Promega; Madison, Wis.).

Protein Assay:

Total protein concentration for all proteins was determined using theMicro BCA (bicinchoninic acid) Assay (Pierce; Rockford Ill.) in themicroplate format, using bovine serum albumin as a standard, accordingto the manufacturer's instructions.

Endotoxin Assay:

Endotoxin levels for all proteins were determined using the QCL-1000Quantitative Chromogenic LAL test kit (Cambrex; E. Rutherford, N.J.),following the manufacturer's instructions for the microplate method.

TLR5 Bioactivity Assay:

HEK293 cells constitutively express TLR5, and secrete several solublefactors, including IL-8, in response to TLR5 signaling. Cells wereseeded in 96-well microplates (50,000 cells/well), and the followingtest proteins were added and incubated overnight: STF2.HA1-1His(PR8)(SEQ ID NO: 89); STF2.HA1-2(PR8) (SEQ ID NO: 90); STF2.HA1-2(PR8)mut(SEQ ID NO: 91); STF2.HA1-3(PR8) SEQ ID NO: 92); HA1-2His(PR8) (SEQ IDNO: 93); STF2.HA1-2 (Mal) (SEQ ID NO: 211) supernatant; STF2. HA1-2(Mal) (SEQ ID NO: 211) refolded; STF2.HA1-2 (SH) (SEQ ID NO: 211)supernatant; STF2.HA1-2 (SH) (SEQ ID NO: 211) refolded; STF2.HA1-1 (Mal)(SEQ ID NO: 209) supernatant; and STF2.HA1-1 (Mal) (SEQ ID NO: 209). Thenext day, the conditioned medium was harvested, transferred to a clean96-well microplate, and frozen at −20° C. After thawing, the conditionedmedium was assayed for the presence of IL-8 in a sandwich ELISA using ananti-human IL-8 matched antibody pair (Pierce, Rockland, Ill.; #M801Eand #M802B) following the manufacturer's instructions. Optical densitywas measured using a microplate spectrophotometer (FARCyte, GE/Amersham;Piscataway, N.J.).

Protein Antigenicity ELISA:

To determine whether the recombinant fusion proteins displayed correctlyfolded epitopes of HA, the antigenicity of individual HA-fusion proteinswas evaluated by ELISA. 96-well ELISA plates were coated overnight at 4°C. with serial dilutions in PBS (100 μl/well) of each target proteinstarting at 5 μg/ml. Plates were blocked with 200 μl/well of AssayDiluent Buffer (ADB; BD Pharmingen) for one hour at room temperature,then washed three times in PBS-T. A fixed dose of primary antibody wasthen added to each well.

To assay HA reactivity, 100 μl/well of a 1:10,000 dilution of non-immuneor PR/8/34 convalescent immune serum in ADB was added. PR/8/34 immuneserum was generated in BALB/c mice (Jackson Laboratory, Bar Harbor, Me.)that received an experimentally determined sublethal challenge dose of8×10¹ egg infectious dosages (EID) of PR/8/34 influenza virus. Animalswere then allowed to convalesce for >21 days post-infection at whichtime immune serum was isolated and clarified. For ELISA of flagellin orthe 6× histidine tag, monoclonal antibody against 6× His (Invitrogen;Carlsbad, Calif.), or flagellin (Inotek; Beverly, Mass.) was added at 1μg/ml in ADB (100 μl/well) and the plates were incubated for 1 hr atroom temperature or overnight at 4° C. The plates were then washed threetimes with PBS-T. HRP-labeled goat anti-mouse IgG antibodies (JacksonImmunochemical; West Grove, Pa.) diluted in ADB were added (100 μl/well)and the plates were incubated at room temperature for 1 hour. The plateswere washed three times with PBS-T. After adding TMB Ultra substrate(Pierce; Rockford, Ill.) and monitoring color development, A₄₅₀ wasmeasured on a microplate spectrophotometer (FARCyte, GE/Amersham;Piscataway, N.J.).

Results and Discussion

Protein Yield and Purity:

Results for the purification of recombinant HA and STF2.HA fusionproteins produced in E. coli are shown in Table 6. All four proteinswere produced in high yield, with estimated purity exceeding 90% andendotoxin well below the standard acceptable level of 0.1 EU/μg. Thethree STF2 fusion proteins also had very high in vitro TLR5 bioactivitywhile the HA1-2His₆ (SEQ ID NO: 93) protein, as expected, had no TLR5activity.

TABLE 6 SEQ purity TLR5 activity ID yield est. endotoxin (EC₅₀, proteinNO: (mg) (%) (EU/μg) ng/ml) STF2.HA1-1His(PR8) 89 6.4 >90 0.04 0.2STF2.HA1-2(PR8) 90 6.0 >98 0.02 0.15 STF2.HA1-2(PR8)mut 91 12 >95 0.011.2 STF2.HA1-3(PR8) 92 2.1 >90 0.016 1.2 HA1-2His(PR8) 93 10 >95 0.03 nd

Antigenicity of Influenza A HA Proteins Produced in E. coli.:

All four recombinant proteins were analyzed by western blotting withantibody against STF2 (flagellin) (Inotek; Beverly, Mass.) and immuneantisera collected from PR/8/34 convalescent mice. The three STF2 fusionproteins appeared to react comparably with anti-flagellin antibody.STF2.HA1-1His(PR8) (SEQ ID NO: 89), STF2.HA1-2(PR8) (SEQ ID NO: 90) andHA1-2His(PR8) (SEQ ID NO: 93) also reacted with anti-PR/8/34convalescent serum. This reactivity was only seen in non-reduced proteinsamples; the addition of DTT to the sample buffer greatly diminished thesignal. This finding suggests that the majority of the anti-HAantibodies in the convalescent serum are conformationally dependent andthat proper disulfide bonding in HA is necessary for reactivity bywestern blot. In contrast to these results, STF2.HA1-3(PR8) (SEQ ID NO:92) did not react efficiently with the convalescent serum and whatlittle signal was produced was not affected by reductant. This findingsuggested that the STF2.HA1-3 protein may not be capable of foldingproperly and/or presenting native HA epitopes.

Capture ELISA for Influenza B Globular Head Proteins

The antigenicity of individual HA-fusion proteins was evaluated usingcapture ELISA. An anti-flagellin monoclonal antibody was used on thesolid phase to capture these proteins. Ferret anti-sera raised onnatural B/Malaysia/2506/2004 infection was used to detect the capturedHA-fusion proteins. The results of this assay will determine whether therecombinant fusion proteins displayed correctly-folded epitopes of HA.Due to the considerable sequence homology between the differentInfluenza B HA globular head molecules, 84.6%, these antisera would beexpected to react with both the Malaysian and the Shanghai globular headconstructs.

Preparation of Samples

E. coli cells expressing the STF2.HA1-2(Malaysia (SEQ ID NO: 210)),STF2.HA1-2(Shanghai (SEQ ID NO: 211)), and STF2.HA1-1 (Malaysia (SEQ IDNO: 209)) recombinant proteins were pelleted by centrifugation andresuspended in Tris buffer (50 mM Tris, 200 mM NaCl, pH 8). Cells werelysed by sonication. The cell lysate was centrifuged to separate thesoluble proteins from the insoluble proteins. The insoluble proteinpellet was resuspended in Tris buffer with 6M urea and the proteins weresubjected to rapid refolding by quickly diluting the solution 1:10 foldin Tris buffer. The protein concentrations of both the soluble proteinsand the pelleted samples containing refolded proteins were estimated byUV₂₈₀ (Spectrophotometer DU 800). Supernatants containingSTF2.HA1-1(Mal) (SEQ ID NO: 209); STF2.HA1-2(Mal) (SEQ ID NO: 210),STF2.HA1-2(SH, Shanghai) (SEQ ID NO: 211) protein and solubilizedpellets containing refolded STF2.HA1-1(Mal), STF2.HA1-2(Mal),STF2.HA1-2(SH) were evaluated by ELISA for reactivity with ferretantisera raised on natural infection against B/Malaysia/2506/2004. E.coli supernatant samples and solubilized, refolded samples containingSTF2.4×M2e (SEQ ID NO: 94) protein were used as negative controls.

ELISA Method

The ELISA plate (Maxisorp, Nunc, Denmark) was coated with theflagellin-specific monoclonal antibody 6H11 (Inotek, Beverly, Mass.) at0.5 ug/mL and incubated overnight at 2-5° C. The antibody coatingsolution was aspirated and the wells were blocked with 300 uL/well SuperBlock+Tween-20 for 2 hours at 25° C. The plate was washed once with1×PBS and blot-dried. Three-fold serial dilutions of the differentprotein solutions were performed in a dilution plate starting with aconcentration of 5 ug/mL. 100 uL was transferred from the dilution plateto the ELISA plate.

The plate was incubated for 1 hr at 25° C. Unbound protein was removedby washing the plate 3 times with PBS+0.05% Tween-20. Ferret antiserumraised against B/Malaysia/2506/2004 (CDC, AL, Georgia) was diluted to1:100 and 100 uL was added to each well. The plate was then incubatedfor 1 hr at 25° C. After this incubation step, the plates were washed 6times with PBS containing 0.05% Tween-20. Goat anti-ferret IgGconjugated to horseradish peroxidase (HRP, Bethyl Labs Inc., IL) wasdiluted 1:10,000 and 100 uL was added to each well. The plate wasincubated for 30 min. After this incubation step, the plate was washed 6times with PBS+0.05% Tween-20. 100 uL of TMB Ultra containing the HRPsubstrate 3,3′,5,5′-tetramethylbenzidine (Pierce, Rockford, Ill.) wasadded to each well. After the addition of this substrate, the colordevelopment was monitored and the reaction was stopped with the additionof 100 uL/well 1M H₂SO₄. A₄₅₀ was measured on a microplate reader(SpectraMax 190, Molecular devices, Sunnyvale, Calif.).

Antigenicity of Influenza B HA Proteins Produced in E. coli

The ELISA data (FIG. 3) indicated that the antisera recognized epitopesthat were sensitive to reductant. Treatment of proteins with reducingagents, which alter the conformation of the proteins by the disruptionof disulphide bonds and, diminished reactivity of the antisera with theproteins. The reactivity of STF2.HA1-2(Mal) total lysate non-reduced,STF2.HA1-2(SH) total lysate non-reduced and STF2.HA1-1 (Mal) totallysate non-reduced when maintained in the properly folded configurationwas very good and indicates that the conformation of the individualHA-fusion proteins was comparable to that of the globular head in thenative HA protein. While the antiserum contains conformationallysensitive antibodies and the reactivity depends on correct disulfidebonding, it also contains antibodies that are non-sensitive to disulfidebonding as demonstrated by the residual activity which was observed inboth reduced and non-reduced conditions.

TLR5Bioactivity of STF2.HA (Influenza B) Proteins

The recombinant fusion proteins showed potency which matched the resultsfrom the Capture ELISA indicating that refolded samples may be moreactive than the lysate in its native form (FIGS. 4 and 5). The samplesin the native untreated form showed activity which were 2-fold less thanthe refolded samples. These proteins were misfolded in the native formand therefore require a refolding step to reinstall the TLR-5bioactivity. This activity was consistent with HA activity in theCapture ELISA.

Example 5 Immunogenicity of Recombinant Flagellin-Hemagglutinin FusionProteins Representing Viral Strain A/PUERTO RICO/8/34 in E. coliMaterials and Methods

Animal Studies:

Female BALB/c mice (Jackson Laboratory, Bar Harbor, Me.)) were used atthe age of 6-8 weeks. Mice were divided into groups of 10 and receivedinguinal subcutaneous (s.c) immunizations on days 0 and 14 as follows:

1) PBS (phosphate buffered saline).

2) 3 μg of STF2.4×M2e (SEQ ID NO: 94) in saline buffer (10 mM Histidine,10 mM Tris, 75 mM NaCl, 5% (vol/vol) sucrose, 0.02% (w/v)Polysorbate-80, 0.1 mM EDTA, 0.5% (v/v) ethanol, pH 7.2)

3) 30 μg of STF2.HA1-2(PR8) (SEQ ID NO: 90) in PBS

4) 3.0 μg of STF2.HA1-2(PR8) (SEQ ID NO: 90) in PBS

5) 0.3 μg of STF2.HA1-2(PR8) (SEQ ID NO: 90) in PBS

6) 0.03 μg of STF2.HA1-2(PR8) (SEQ ID NO: 90) in PBS

An additional group of five mice received an experimentally determinedsublethal challenge with 8×10¹ egg infectious dosages (EID) PR/8/34 andwere allowed to convalesce for >21 days. These animals were then used asimmune convalescent positive controls during the challenge studies (seebelow). Mice were bled on days 10 (primary) and 21 (boost), and serawere clarified by clotting and centrifugation and stored at −20° C.

Serum Antibody Determination:

HA-specific IgG levels were determined by ELISA. 96-well ELISA plates(Costar (Cat #9018) Corning, N.Y.) were coated overnight at 4° C. with100 μl/well HA0sHis protein (produced in Drosophila) (SEQ ID NO: 176) inPBS (5 μg/ml). Plates were blocked with 200 μl/well of Assay DiluentBuffer (ADB; BD Pharmingen, (Cat#: 555213) (San Diego, Calif.) for onehour at room temperature. The plates were washed three times inPBS+0.05% (v/v) Tween 20 (PBS-T). Dilutions of the sera in ADB wereadded (100 μl/well) and the plates were incubated overnight at 4° C. Theplates were washed three times with PBS-T. HRP-labeled goat anti-mouseIgG antibodies (Jackson Immunochemical, West Grove, Pa. (Cat#:115-035-146)) diluted in ADB were added (100 μl/well) and the plateswere incubated at room temperature for 1 hour. The plates were washedthree times with PBS-T. After adding TMB Ultra substrate (Pierce (Cat34028), Rockford, Ill.)) and monitoring color development, A₄₅₀ wasmeasured on a Tecan Farcyte (Durham, N.C.) microplate spectrophotometer.

MDCK Whole Cell ELISA:

MDCK cells (ATCC (Cat#CCL-34) Manassas, Va.) were grown in 96-wellculture plates (BD (Cat 353075), Corning, N.Y.) in DMEM complete mediumcontaining 10% FCS at 37° C. for one to two days or until cells werenear confluence. Wells were then incubated with 1×10⁶ EID of PR/8/34virus (50 μl) in DMEM medium without FCS or medium alone (for uninfectedcontrols).

Following a 60 minute incubation at 37° C., 200 μl of complete mediumwas added to each well and plates were incubated overnight at 37° C. Thenext day plates were washed with PBS and fixed with 10% formalin at roomtemperature for 10 minutes. Wells were washed three times with PBS/0.1%BSA and blocked with 200 μl/well ADB (BD Pharmingen, Cat#555213), SanDiego, Calif.) for one hour at RT or overnight at 4° C. Serial dilutionsof test sera were added to the wells and incubated for one to two hoursat room temperature. Wells were washed and incubated with HRP-conjugatedgoat anti-mouse IgG (Jackson Immunochemical, Cat 115-035-146 (WestGrove, Pa.) for 30 minutes at room temperature, followed by TMB Ultrasubstrate (Pierce (Cat#34028), Rockford, Ill.) for two minutes at roomtemperature. The reaction was stopped with the addition of 25 μl of 1NH₂SO₄ and the OD₄₅₀ was read using a microplate spectrophotometer(FARCyte, Amersham, Durham, N.C.).

Results and Discussion

Induction of HA-specific IgG Response Following Immunization withSTF2.HA1-2(PR8) (SEQ ID NO: 90):

The immunogenicity of STF2.HA1-2 was examined by immunizing BALB/c mice(10/group) subcutaneously on day 0 and 14 with a dose range ofSTF2.HA1-2 (30, 3.0, 0.3, or 0.03 μg). Control groups of mice wereimmunized with PBS (negative control), 3 μg of STF2.4×M2e (SEQ ID NO:94) (negative control for HA immunogenicity, positive control for lethalchallenge efficacy study), or a sub-lethal challenge with 8×10¹ EID ofthe influenza isolate PR/8/34 to generate immune convalescent animals.HA-specific IgG responses were examined 7 days post boost (Day 21) byELISA. The results demonstrate that immunization with 30, 3 or 0.3 μg ofSTF2.HA1-2(PR8) induced consistent and significant HA0sHis-specific (SEQID NO: 176) IgG responses in a dose-dependent manner (FIG. 6).

Sera from BALB/c Mice Immunized with STF2.HA1-2 (SEQ ID NO: 90) Reactwith MDCK Cells Infected with Influenza Virus:

The direct ELISA results above demonstrated that the immune sera fromSTF2.HA1-2-immunized animals recognize recombinant Drosophila expressedHA0sHis (SEQ ID NO: 176) corresponding to the PR/8/34 HA sequence. Inorder to demonstrate that the anti-HA antibodies recognize native viralHA, the same sera were examined for the ability to react with MDCK cellsinfected with PR/8/34. The results shown in FIG. 7 demonstrate that serafrom mice immunized with 30, 3.0, or 0.30 μg of STF2.HA1-2 boundspecifically to PR/8/34-infected MDCK cells, indicating that the anti-HAantibodies elicited by immunization with STF2.HA1-2 recognize HA in itsnative conformation.

Collectively these results demonstrate that mice immunized withSTF2.HA1-2(PR8) (SEQ ID NO: 90) in PBS, without conventional adjuvant orcarrier, mounted vigorous anti-HA responses that recognized recombinantHA expressed in Drosophila and native viral HA expressed on the surfaceof PR/8/34 infected MDCK cells in vitro.

Example 6 Efficacy of Recombinant Flagellin-Hemagglutinin FusionProteins Representing Viral Strain A/PUERTO RICO/8/34 Materials andMethods

Influenza Virus Challenge of Mice.

To assess efficacy, mice immunized as described in Example 5 werechallenged on day 28 by intranasal administration of an LD₉₀ (doselethal to 90% of mice)(8×10³ EID) of influenza A isolate PR/8/34.Animals were monitored daily for 21 days following the challenge forsurvival, weight loss and clinical presentation. The % weight loss wascalculated based on the mean of ((Daily weight (g)/Initial weight (g)day 28)×100) of each individual animal per group. Clinical scores wereassigned as follows: 4 pts=healthy, 3 pts=reduced grooming, 2pts=reduced physical activity and 1 pt=moribund. (Experimental resultsfor clinical scores and weight loss reflect the results based onsurviving animals on the day evaluated).

Virus Titration and Determination of TCID₅₀.

Cell Preparation:

MDCK cells (ATCC (Cat#CCL-34), Manassas, Va.) were cultured in 100×20 mmculture plates (BD (Cat#353003) Corning, N.Y.) to 90-95% confluency andthe monolayer was dislodged by incubation with trypsin-EDTA at 37° C./5%CO₂ for 20 minutes. The trypsin was inactivated by the addition of DMEMcell culture medium supplemented with 10% FBS, and the cell monolayerwas scraped with a sterile spatula to complete detachment of cells. Thecell suspension was harvested and washed twice with DMEM+10% FBS. Cellswere resuspended in DMEM+10% FBS and counted. The cell concentration wasadjusted to 4×10⁵ cells/ml and 100 μl was added to each well of a96-well tissue culture plate (BD (Cat#353075), Corning N.Y.). Plateswere incubated at 37° C./5% CO₂ until confluence reached 90-95%.

Viral Titration:

Influenza virus (strain A/Puerto Rico/8/34 [PR/8]) was diluted to 1×10⁸EID in phenol red-free DMEM+0.1% BSA (fractionV (Rockland (Cat#BSA-50)Gilbertsville, Pa.), and serial 5-fold dilutions were prepared in96-well plates using the same medium. Monolayers of MDCK cells in96-well plates, prepared as described in herein, were washed byaspirating the culture medium, replacing with 200 μl/well of 1×PBS, andaspirating the PBS. Serial 5-fold dilutions of influenza virus preparedabove were added to the washed monolayers in a volume of 100 μl/well.One row of wells was treated with medium only as a control. The cellswere incubated for 2 hours at 37° C./5% CO₂ to allow viral attachmentand entry. Wells were washed by aspiration, rinsing with 200 μl/wellPBS, and aspiration of the PBS. All wells received 100 μl/well phenolred-free DMEM+0.1% BSA and the plates are incubated for 48 hours at 37°C./5% CO₂.

Determination of Cell Viability:

Following incubation with virus as described above, the medium wasaspirated from the wells and replaced with fresh medium containing 40μg/ml neutral red (Sigma Aldrich (Cat#N2889) St. Louis, Mo.). Todetermine maximum lysis, 2 μl lysis solution (9% Triton X-100 in water,weight/vol) was added to triplicate wells that were incubated withmedium only. Following a one hour incubation, the cells were fixed bythe addition of 100 μl/well 1% formaldehyde/1% CaCl₂ for 5 minutes atroom temperature; this fix step was performed twice in succession. Thefix solution was aspirated and the neutral red was released by theaddition of 100 μl/well of extraction medium (50% ethanol/1% aceticacid).

The plate was incubated at room temperature for 20 minutes, with shakingfor the final 2 minutes. The amount of dye released was determined bymeasuring absorbance at a wavelength of 540 nm using a microplatespectrophotometer. Cell death (and hence, viral infectivity) wasmeasured as a decrease in the amount of dye released as compared tomedia control. At the end of the incubation, the neutral red assay wasperformed as described herein. The percentage lysis of each serumdilution was calculated as:% reduction=100×((sample-virus)/(med-virus))where sample, max, and med refer to the absorbance values in wellsrepresenting experimental samples, virus only, and medium only,respectively. The neutralizing titer of each sample was defined as thedilution of serum which resulted in a 50% reduction in viralinfectivity.

Influenza A/PR/8/34 Neutralization Assay

This assay was adapted with modifications from WHO Manual on AnimalInfluenza Diagnosis and Surveillance, p. 86-88 (WHO/CDS/CSR/NCS2002.5).The Neutral Red Assay is adapted from a public protocol of the Cell Labat Gettysburg College.

Specifically, MDCK (ATCC, Cat#(CCL_(—)34), Manassas, Va.) cells wereplated in 96-well tissue culture plates (BD (Cat 353075), Corning, N.Y.)as described above. Test reagents (experimental and control sera) wereheat-inactivated by incubating for 30 minutes in a water bath heated to56° C. Sera were serially titrated in 96-well plates, in 3-folddilutions in phenol red-free DMEM+0.1% BSA. An equal volume of PR/8/34virus diluted to 5×10⁶/ml EID in the same medium was added to each serumdilution to achieve a final viral concentration of 2.5×10⁶ EID/ml (Thepre-determined TCID₅₀ for our current stock of virus). Wells containingmedium only and virus only were included as negative and positivecontrols, respectively. The plates were incubated for 30 minutes at 37°C./5% CO₂.

Cell monolayers prepared as described above (Cell preparation) werewashed once with 200 μl/well 1×PBS, then 100 μl/well of serum:virusmixtures and control reagents prepared as described above were added andincubated for 2 hours at 37° C./5% CO₂. Following incubation, cellmonolayers were washed once with PBS, and 100 μl/well phenol red-freeDMEM+0.1% BSA was added to each well and incubated at 37° C./5% CO₂ for48 hours. At the end of the incubation, the neutral red assay wasperformed as described above (Determination of cell viability). Thepercentage lysis of each serum dilution was calculated as describedabove. The neutralizing titer of each sample was defined as the dilutionof serum which results in a 50% reduction in viral infectivity

Results and Discussion

Immunization with STF2.HA1-2(PR8) (SEQ ID NO: 90) Provides Protectionfrom a Lethal Challenge with Influenza A:

The results from Example 5 demonstrated that immunization of mice withSTF2.HA1-2(PR8) (SEQ ID NO: 90) generated an antibody response thatrecognized native HA. In order to evaluate efficacy, the same mice werechallenged on day 28 with an LD₉₀ (8×10³EID) of PR/8/34 virusadministered intra-nasally. Mice were monitored daily for 19 daysfollowing the challenge for survival, weight loss and clinicalpresentation. As shown in FIGS. 8A, 8B, and 8C, PBS-immunized miceshowed signs of infection (weight loss and lower clinical scores) asearly as three days post-challenge and all mice died by day 21post-challenge. In contrast, mice immunized with 30, 3.0 or 0.3 μg ofSTF2.HA1-2(PR8) (SEQ ID NO: 90) demonstrated markedly enhancedprotection. These animals demonstrated little to no weight loss,significantly higher clinical scores and 100% survival that was similarto immune convalescent control animals over the 21 day period. Theseresults demonstrate that E. coli-expressed STF2.HA1-2(PR8) (SEQ ID NO:90) induces HA-specific immune responses that successfully protectBALB/c mice from a lethal challenge with virulent influenza A virus invivo.

In order to evaluate the effectiveness of the antibody response in vitroa cell-based virus neutralization assay was developed to test theability of immune sera to neutralize viral infectivity. In this studythe viral inhibitory activity of serum from animals immunized withSTF2.HA1-2(PR8) (SEQ ID NO: 90) and STF2.HA1-1(PR8) (SEQ ID NO: 151) wasexamined. Serial dilutions of non-immune and immune sera werepre-incubated with PR/8 and incubated with MDCK cells. Wells were washedto remove free virus and plates were incubated prior to staining withneutral red to detect live cells. The results indicate that immune serafrom animals immunized with STF2.HA1-2(PR8) (SEQ ID NO: 90) demonstrateda tissue culture inhibition 50% dose (TCID) of >1:40 (FIG. 9).

Example 7 Immunogenicity of Recombinant Flagellin-Hemagglutinin FusionProteins Representing Viral Strain A/Viet Nam/1203/2004 Materials andMethods

Animal Studies:

Female BALB/c mice (Jackson Laboratory, Bar Harbor, Me.) were used atthe age of 6-8 weeks. Mice were divided into groups of 10 and receivedinguinal s.c immunizations on days 0 and 14 as follows:

1) PBS (phosphate buffered saline).

2) 3.0 μg of STF2.HA1-2(PR8) (SEQ ID NO: 90) in PBS

3) 3.0 μg of STF2.HA1-2(VN) (SEQ ID NO: 95) in PBS

4) 0.3 μg of STF2.HA1-2(VN) (SEQ ID NO: 95) in PBS

Mice were bled on day 21 and sera were clarified by clotting andcentrifugation and stored at −20° C.

Serum Antibody Determination:

HA and STF2-specific IgG levels were determined by ELISA. 96-well ELISAplates (Costar (Cat#9018), Corning, N.Y.) were coated overnight at 4° C.with 100 μl/well of HA protein purified from Viet Nam/1203/2004 (BEIResources (Cat #NR-660), Manassas, Va.)) or recombinant flagellin (STF2)(SEQ ID NO: 96) in PBS (5 μg/ml). Plates were blocked with 200 μl/wellof Assay Diluent Buffer (ADB; BD Pharmingen (Cat#555213) San Diego,Calif.) for one hour at room temperature. The plates were washed threetimes in PBS buffer containing 0.05% (v/v) Tween 20 (PBS-T). Dilutionsof the sera in ADB were added (100 μl/well) and the plates wereincubated overnight at 4° C. The plates were washed three times withPBS-T. HRP-labeled goat anti-mouse IgG antibodies (JacksonImmunochemical (Cat#115-035-146), West Grove, Pa.) diluted in ADB wereadded (100 μl/well) and the plates were incubated at room temperaturefor 1 hour. The plates were washed three times with PBS-T. After addingTMB Ultra substrate (Pierce (Cat#34028), Rockford, Ill.)) and monitoringcolor development, A₄₅₀ was measured on a Tecan Farcyte (Durham, N.C.)microplate spectrophotometer.

Results and Discussion

The immunogenicity of STF2.HA1-2(VN) (SEQ ID NO: 95) was examined inBALB/c mice. Animals were immunized s.c. on days 0 and 14 with 3 μgSTF2.HA1-2(PR8) (SEQ ID NO: 90), or 3 or 0.3 μg of STF2.HA1-2(VN) (SEQID NO: 95). On day 21 animals were bled and serum IgG responses topurified HA from A/Viet Nam/1203/2004 (obtained from BEI ResourcesCat#NR-660) and recombinant flagellin (STF2; (SEQ ID NO: 96)) wereexamined by ELISA (FIGS. 10A and 10B). The results demonstrate thatserum from mice immunized with STF2.HA1-2(VN) (SEQ ID NO: 95) exhibitantigen-specific reactivity with purified H5 protein from influenzaA/Viet Nam/1203/2004.

Example 8 Cloning, Expression, and Biochemical Characterization ofRecombinant Flagellin-Hemagglutinin Fusion Proteins Produced inBaculovirus

Cloning:

Expression of HA and STF2.HA fusion proteins was carried out using theBac-to-Bac Baculovirus Expression System (Invitrogen, Carlsbad, Calif.)according to the manufacturer's guidelines (FIG. 11). cDNAs encoding HAand STF2.HA fusion proteins were cloned into the pFastBac donor plasmidwhich was then used to transform DH10Bac cells containing the bacmid andhelper DNA. Recombinant bacmid clones generated by homologousrecombination in DH10Bac were then identified by blue-white screening onX-gal plates. The details of the cloning procedure are given in thefollowing paragraphs.

Generation of Vector Cassettes:

The pFastBac™1 vector is compatible with the Bac-to-Bac® BaculovirusExpression System (Invitrogen, Carlsbad, Calif.). This vector has astrong AcMNPV polyhedrin (PH) promoter for high level protein expressionand a large multiple cloning site for simplified cloning. pFastBac™1 isa non-fusion vector (i.e. no fusion tags are present in the vector).

To ensure proper expression of recombinant protein, the insert mustcontain an ATG start codon for initiation of translation and a stopcodon for termination of translation. In order to facilitate cloning offlagellin in fusion with several truncations of the HA gene, two plasmidcassettes were engineered: pFB-STF2B1p.wt (SEQ ID NO: 97) andpFB-STF2B1p.ng (SEQ ID NO: 98). In the latter, the putativeglycosylation sites in the flagellin (STF2) gene were obliterated bysubstituting Gln (Q) for Asn (N). Potential glycosylation sites weredetermined using the consensus sequence for N-glycosylation N-X-S/T: Nresidue followed by any residue by S or T in the third position.

In the certain locations, residues 40, 122, 215, 237, 414, 478, and 497of the STF2 gene (SEQ ID NO: 212) the N residue was incorporated as a Qsubstitution. Constructs which harbor these mutations and in which theproteins are not likely to be modified by sugars are designated asnon-glycosylated (ng) mutants. Similarly, glycosylation mutations wereintroduced in HA0s gene at positions 11, 23, 268, 286 and 480 (SEQ IDNO: 23). Both vector constructs (pFB-STF2B1p.wt and pFB-STF2B1p.ng)harbor a silent mutation at nucleotides 5′-GTGCTGAGCCTGTTACGT-3′ (SEQ IDNO: 310) (nt 1501 to 1518) of STF2 (SEQ ID NO: 212) creating a uniqueBlpI in the plasmid cassette. Each cassette contains the honey beemelittin (HBM) sequence (SEQ ID NO: 99) fused at the amino terminus ofSTF2 to provide a signal for secretion. Both plasmids werecodon-optimized for expression in Baculovirus (Midland Certified ReagentCo., Inc, Midland, Tex.; DNA2.0 Inc, Menlo Park, Calif.). The syntheticgenes were excised with BamHI or BglII and SphI and cloned intopFastBac™1 vector by compatible end ligation generating thepFB-STF2B1p.wt and pFB-STF2B1p.ng cassettes.

Flagellin-HA Fusion Constructs:

Subunits of the HA globular head from influenza strains A/PuertoRico/8/34, A/Viet Nam/1203/2004, A/Indonesia/2005 and A/NewCaledonia/12/99 were expressed alone or genetically fused to flagellin(STF2) (SEQ ID NO: 178) and expressed in Baculovirus. This wasaccomplished by one of three methods.

Method #1:

To generate reagents for ELISA, HA1-1 subunits of PR8, VN, IND and NCstrains (Tables 7 and 8) were cloned into pFASTBac™1, generatingpFB.HA1-1 that harbors a hexa-his tag. This was achieved by employingPCR with a set of primers as indicated in the Tables below and with asynthetic codon-optimized HA0s gene (HA excluding the signal sequenceand extracellular domain) as DNA template (DNA2.0 Inc., Menlo Park,Calif.). The PCR fragments were digested with BglII and SphI andinserted into pFastBac™1 that has previously been treated with BglII andSphI enzymes followed by BAP treatment. In order to generate aflagellin-HA subunit fusion, the PCR product was digested with BlpI, andligated by compatible ends to pFB-STF2B1p.wt.

Method #2:

In this protocol, codon-optimized HA subunit genes representing both thewt and non-glycosylated forms were chemically synthesized (DNA2.0 Inc.,Menlo Park, Calif.). The genes were excised with BlpI and SphI enzymesand the fragment gel purified and ligated to pFB-STF2B1p.wt or thenon-glycosylated version, pFB-STF2B1p.ng previously digested with BlpIand SphI and BAP treated. In each case, the ligation mix was used totransform TOP10 cells and transformants were screened by PCR and DNAsequencing to confirm the presence and correct orientation of theinserts. The constructs were used to transform MAX Efficiency® DH10Bac™competent E. coli (Invitrogen, Carlsbad, Calif.) to generate arecombinant bacmid. The colonies of bacteria were screened for positivebacmids by blue/white selection on plates containing 50 μg/mL kanamycin,7 μg/mL gentamycin, 10 μg/mL tetracycline, 40 μg/mL IPTG and Bluo-Gal(40 μg/mL). Recombinant bacmid DNA was prepared and used to transfectthe insect cell line of choice (Sf9 or Sf21 cells) to generate arecombinant Baculovirus. The baculoviral stock was then amplified andtitered and used to infect High Five insect cells to express therecombinant protein.

TABLE 7 PR8 HA constructs for expression in Baculovirus FOR REV DNA SEQPrimer Primer Template ID Construct SEQ ID SEQ ID SEQ ID NO: (His tag)Method NO: NO: NO: 100 STF2.HA0s #1 101 102 103 104 STF2.HA1-1 #1 105106 103 107 STF2.HA1-2 #1 108 109 103 110 STF2.HA1-2mut #2 N/A N/A 111112 STF2.HA1-3 #1 113 114 103 115 STF2.HA1-3mut #2 N/A N/A 116 117ngSTF2.HA0s #2 N/A N/A 118 119 ngSTF2.HA1-1 #2 N/A N/A 120 121ngSTF2.HA1-2 #2 N/A N/A 122 123 ngSTF2.HA1-2mut #2 N/A N/A 124 125ngSTF2.HA1-3 #2 N/A N/A 126 127 ngSTF2.HA1-3mut #2 N/A N/A 128 129wtSTF2.HA1-1ng #2 N/A N/A 130 131 ngSTF2.HA1-1wt #2 N/A N/A 132 133HA1-1 #1 134 135 136 137 HA1-1 (no tag) #1 134 138 136

TABLE 8 NC, VN and NC HA constructs for expression in Baculovirus FORREV DNA SEQ Primer Primer Temlate ID Construct SEQ ID SEQ ID SEQ ID NO:(his tag) Method NO: NO: NO: 139 HA1-1(NC) #1 140 141 142 143 HA1-1(VN)#1 144 145 146 147 HA1-1(IND) #1 148 149 150

Protein Expression:

Spodoptera frugiperda (Sf9) insect cells were cultured in Grace's InsectCell Medium (Invitrogen; Carlsbad, Calif.). Recombinant bacmids werepurified from DH10Bac clones and transfected into Sf9 cells usingCellfectin (Invitrogen; Carlsbad, Calif.) to generate passage one (P1)Baculovirus stocks which were then titered by traditional plaque assayaccording to the manufacturer's guidelines. Subsequent infections of Sf9cells were performed using P1 viral stocks at a multiplicity ofinfection (MOI) of 0.1 to produce larger volume P2 viral stocks withincreased viral titers. Cultures expressing the following proteins werescaled up for protein purification: ngSTF2.HA(PR8)1-2mutHis (SEQ ID NO:156); ngSTF2.HA(PR8)1-1His (SEQ ID NO: 152; STF2.HA(PR8)1-1His (SEQ IDNO: 151); STF2.HA(PR8)1-2His (SEQ ID NO: 153); STF2.HA1-2mutHis (SEQ IDNO: 155); HA(PR8)1-1His (SEQ ID NO: 179) HA(VN)1-1His (SEQ ID NO: 180);HA(IND)1-1His (SEQ ID NO: 181); HA(NC)1-1His (SEQ ID NO: 182). Proteinexpression was performed by infecting 2×1 L flasks of High-5 insectcells with P2 virus at an MOI of 2 and culturing for 24 hrs at 28° C.with slow shaking Conditioned medium was harvested by centrifugation andclarified by passing through a 0.22 μm filter and stored at 4° C.

Protein Purification:

NiSO₄ was added to each clarified supernatant to a final concentrationof 0.5 mM and the pH adjusted to 8.0. The His₆-tagged protein was thencaptured on a chelating sepharose column (GE/Amersham Biosciences;Piscataway, N.J.) charged with NiSO₄ and equilibrated in Buffer A (20 mMTris, pH 8.0, 0.5 M NaCl). After washing with Buffer A the bound proteinwas eluted with Buffer B (Buffer A+0.5 M imidazole). Peak fractions werepooled, dialyzed overnight into Buffer A and applied to a nickel NTAcolumn (Sigma-Aldrich; St. Louis, Mo.). The column was eluted with a5-column volume gradient from 100% Buffer A to 100% Buffer B. Peakfractions were pooled and dialyzed into 1×TBS, pH 8.0.

SDS-PAGE and Western Blot Analysis:

Protein identity was determined, and purity estimated, by SDS-PAGE. Analiquot of 5 μg of each sample was diluted in SDS-PAGE sample bufferwith or without 100 mM DTT as a reductant. The samples were boiled for 5minutes and loaded onto a 10% SDS polyacrylamide gel (LifeGels; French'sForrest, New South Wales, AUS) and electrophoresed. The gel was stainedwith Coomassie R-250 (Bio-Rad; Hercules, Calif.) to visualize proteinbands. For western blot, 0.5 μg/lane total protein was electrophoresedas described above and the gels were then electro-transferred to a PVDFmembrane and blocked with 5% (w/v) dry milk before probing withanti-flagellin antibody (Inotek; Beverly, Mass.), anti-His₆ antibody(Invitrogen; Carlsbad, Calif.), or influenza A PR/8/34 convalescentimmune serum (described below under Protein Antigenicity ELISA). Afterprobing with alkaline phosphatase-conjugated secondary antibodies(Pierce; Rockford, Ill.), protein bands were visualized with an alkalinephosphatase chromogenic substrate (Promega; Madison, Wis.).

Protein Assay:

Total protein concentration was determined using the Micro BCA(bicinchoninic acid) Assay (Pierce; Rockford, Ill.) in the microplateformat, using bovine serum albumin as a standard, according to themanufacturer's instructions.

Endotoxin Assay:

Endotoxin levels were determined using the QCL-1000 QuantitativeChromogenic LAL test kit (Cambrex; E. Rutherford, N.J.), following themanufacturer's instructions for the microplate method.

TLR5 Bioactivity Assay:

HEK293 cells constitutively express TLR5, and secrete several solublefactors, including IL-8, in response to TLR5 signaling. Cells wereseeded in 96-well microplates (50,000 cells/well), and the followingtest proteins were added and incubated overnight:ngSTF2.HA(PR8)1-2mutHis (SEQ ID NO: 156); ngSTF2.HA(PR8)1-1His (SEQ IDNO: 152); STF2.HA(PR8)1-1His (SEQ ID NO: 151); STF2.HA(PR8)1-2His (SEQID NO: 153); STF2.HA1-2(PR8)mutHis (SEQ ID NO: 155) The next day, theconditioned medium was harvested, transferred to a clean 96-wellmicroplate, and frozen at −20° C. After thawing, the conditioned mediumwas assayed for the presence of IL-8 in a sandwich ELISA using ananti-human IL-8 matched antibody pair (Pierce; Rockford, Ill., #M801Eand #M802B) following the manufacturer's instructions.

Protein Antigenicity ELISA:

The following constructs purified from Baculovirus culture wereevaluated by ELISA to determine whether the proteins displayed correctlyfolded epitopes of HA: ngSTF2.HA(PR8)1-2mutHis (SEQ ID NO: 156);ngSTF2.HA(PR8)1-1His (SEQ ID NO: 152); STF2.HA(PR8)1-1His (SEQ ID NO:151); STF2.HA(PR8)1-2His (SEQ ID NO: 153); STF2.HA1-2mutHis (SEQ ID NO:155); and HA(PR8)1-1His (SEQ ID NO: 179). 96-well ELISA plates werecoated overnight at 4° C. with serial dilutions in PBS (100 μl/well) ofeach target protein starting at 5 μg/ml. Plates were blocked with 200μl/well of Assay Diluent Buffer (ADB; BD Pharmingen) for one hour atroom temperature, then washed three times in PBS-T. A fixed dose ofprimary antibody was then added to each well. To assay HA reactivity,100 μl/well of a 1:10,000 dilution of non-immune or PR/8/34 convalescentimmune serum in ADB was added. PR/8/34 immune serum was generated inBALB/c mice (Jackson Laboratory, Bar Harbor, Me.) that received anexperimentally determined sublethal challenge dose of 8×10¹ egginfectious dosages (EID) of PR/8/34 influenza virus. Animals were thenallowed to convalesce for >21 days post-infection at which time immuneserum was isolated and clarified.

For ELISA of flagellin or the 6× histidine tag, monoclonal antibodyagainst 6× His (Invitrogen; Carlsbad, Calif.), or flagellin (Inotek;Beverly, Mass.) was added at 1 μg/ml in ADB (100 μl/well) and the plateswere incubated for 1 hr at room temperature or overnight at 4° C. Theplates were then washed three times with PBS-T. HRP-labeled goatanti-mouse IgG antibodies (Jackson Immunochemical; West Grove, Pa.)diluted in ADB were added (100 μl/well) and the plates were incubated atroom temperature for 1 hour. The plates were washed three times withPBS-T. After adding TMB Ultra substrate (Pierce; Rockford, Ill.) andmonitoring color development, A₄₅₀ was measured on a microplatespectrophotometer (FARCyte, GE/Amersham; Piscataway, N.J.).

Results and Discussion

Expression and Purification of Recombinant HA Proteins:

The results of Baculovirus expression of recombinant STF2.HA fusionproteins are summarized in Table 9. All proteins were expressed inmoderate to high yield. Purity after metal chelate chromatography wasgenerally good and endotoxin levels were far below the acceptable limitof 0.1 EU/μg. All STF2 fusion proteins demonstrated potent in vitro TLR5activity.

TABLE 9 SEQ Purity TLR activity ID Yield est. Endotoxin (EC₅₀, ProteinNO: (mg) (%) (EU/μg) ng/ml) STF2.HA1- 151 12 >80 0.01 3 1His(PR8)ngSTF2.HA1- 152 24 >90 <0.01 30 1His(PR8) STF2.HA1- 153 13.2 >90 <0.01 92His(PR8) STF2.HA1- 155 25 >95 <0.01 900 2mutHis(PR8) ngSTF2.HA1- 1566 >90 0.01 9 2mutHis(PR8)

STF2.HA proteins for Baculovirus expression were engineered with andwithout N-linked glycosylation sites in order to determine the effect ofthis posttranslational modification on protein expression, folding, andbiological activity. ngSTF2.HA1-2.mutHis(PR8) (SEQ ID NO: 156), which isnon-glycosylated, appears to be expressed at a lower level inBaculovirus supernatants than the corresponding glycosylated protein,suggesting that glycosylation may influence the folding or secretion ofthis protein. However, the glycosylated and non-glycosylated versions ofthis protein appear to react equally with convalescent antiserum frominfluenza PR8-infected mice when analyzed by western blot. Furthermore,ngSTF2.HA1-1His(PR8) (SEQ ID NO: 152) expressed at higher levels thanits glycosylated counterpart, indicating that any effect ofglycosylation on expression of these proteins may not be easilygeneralized.

In addition to the proteins expressed and purified at large scale,small-scale Baculovirus P1 supernatants of STF2.HA1-3His(PR8) (SEQ IDNO: 157) and STF2.HA1-3mutHis(PR8) (SEQ ID NO: 158) were analyzed for HAantibody reactivity. As seen with the STF2.HA1-3(PR8) protein (SEQ IDNO: 92) expressed in E. coli (See Example #4), these proteins showedvery poor reactivity with PR8 convalescent mouse serum on western blotsof conditioned medium. This result further confirms that STF2.HA1-3proteins, whether expressed in a prokaryotic or eukaryotic host, areunable to fold properly and display native epitopes.

Antigenicity:

Several STF2.HA proteins produced in Baculovirus and E. coli wereanalyzed by ELISA. All STF2 fusion proteins reacted equally well withanti-flagellin antibody (Inotek; Beverly, Mass.), demonstrating equalprotein loading in the assay. Proper folding and display of antibodyepitopes in STF2.HA fusion proteins was then examined by measuringreactivity of each protein with antisera from mice which had recoveredfrom PR/8/34 influenza infection. Reactivity with PR/8/34 convalescentmouse serum was equivalent for Baculovirus-expressed STF2.HA1-1His(PR8)(SEQ ID NO: 151), ngSTF2.HA1-1His(PR8) (SEQ ID NO: 152), andngSTF2HA1-2mutHis(PR8) (SEQ ID NO: 156), and E. coli-expressedSTF2.HA1-2(PR8) (SEQ ID NO: 90) (see Example #4). Thus, serum antibodyrecognition of the HA head domain may be focused on a region encompassedby the HA1-2 construct. Further, in vitro refolding of STF2.HA1-2 (SEQID NO: 90) produced in E. coli results in a protein with equivalentimmune reactivity (and therefore similar folded conformation) to aprotein processed and secreted from a eukaryotic host (Baculovirus). Inaddition, serum antibody recognition of the HA head domain may notappear to be dependent on glycosylation and, at least for PR/8/34 HA,may not be negatively affected by glycosylation. HAs (hemaglutinins)from different influenza strains have variable patterns of N-linkedglycosylation. (glycosylation may negatively affect immune recognitionof other HAs. Serum antibody recognition of HA may be unaffected byN-glycosylation. It is possible glycosylation may otherwise influencethe effectiveness, such as the half-life of a vaccine.

Example 9 Immunogenicity and Efficacy of RecombinantFlagellin-Hemagglutinin Fusion Proteins Produced in Baculovirus Materialand Methods

Animal Studies:

Female BALB/c mice (Jackson Laboratory, bar Harbor, Me.) were used atthe age of 6-8 weeks. Mice were divided into groups of 10 and receivedinguinal subcutaneous immunizations on days 0 and 14 as follows:

1) PBS (phosphate buffered saline).

2) 3 μg of STF2.HA1-2(PR8) E. coli (SEQ ID NO: 90) in PBS

3) 3 μg of STF2.HA1-1(PR8) Baculovirus (SEQ ID NO: 151) in PBS

4) 3 μg of ngSTF2.HA1-1(PR8) Baculovirus (SEQ ID NO: 152) in PBS

5) 0.3 μg of ngSTF2.HA1-1(PR8) Baculovirus (SEQ ID NO: 152) in PBS

6) 3 μg of STF2.HA1-1(PR8) E. coli (SEQ ID NO: 89) in PBS

7) 0.3 μg of STF2.HA1-1(PR8) E. coli (SEQ ID NO: 89) in PBS

Mice were bled on days 10 (primary) and 21 (boost), and sera wereclarified by clotting and centrifugation and stored at −20° C. Immunizedanimals were challenged on day 28 with an LD₉₀ (8×10³ EID) of PR/8/34virus administered intranasally (See Example 6). Mice were monitoreddaily for 21 days following the challenge for survival, weight loss andclinical presentation.

Serum Antibody Determination:

HA-specific IgG levels were determined by ELISA. 96-well ELISA plates(Costar (Cat#9018), Corning, N.Y.) were coated overnight at 4° C. with100 μl/well HA0sHis protein produced in Drosophila (SEQ ID NO 176) inPBS (5 μg/ml). Plates were blocked with 200 μl/well of Assay DiluentBuffer (ADB; BD Pharmingen (Cat#555213), San Diego, Calif.) for one hourat room temperature. The plates were washed three times in PBScontaining 0.05% Tween-20 (PBS-T). Dilutions of the sera in ADB wereadded (100 μl/well) and the plates were incubated overnight at 4° C. Theplates were washed three times with PBS-T. HRP-labeled goat anti-mouseIgG antibodies (Jackson Immunochemical (Cat#115-035-146)) diluted in ADBwere added (100 μl/well) and the plates were incubated at roomtemperature for 1 hour. The plates were washed three times with PBS-T.After adding TMB Ultra substrate (Pierce (Cat#34028), Rockford, Pa.) andmonitoring color development, A₄₅₀ was measured on a Tecan Farcyte(Durham, N.C.) microplate spectrophotometer.

Results and Discussion

Immunogenicity and Efficacy of STF2.HA1-1(PR8) Expressed in E. coli andBaculovirus:

BALB/c mice were immunized s.c. with 3.0 or 0.3 μg of indicatedrecombinant fusion proteins on days 0 and 14. On day 21 mice were bledand HA-specific IgG titers were examined by ELISA against HA0sHis (SEQID NO 176) expressed in Drosophila. FIGS. 12A,12B shows the proteinsinduced varying levels of HA-specific IgG with ngSTF2.HA1-1(PR8) (SEQ IDNO: 152) inducing the strongest response similar to that observed inanimals immunized with STF2.HA1-2(PR8) expressed in E. coli (SEQ ID NO:90). Interestingly, animals immunized with STF2.HA1-1(PR8) containingthe intact glycosylation sequences expressed in Baculovirus (SEQ ID NO:151) elicited little to no detectable antibody responses to HA orflagellin, in marked contrast to that observed in animals similarlyimmunized with STF2.HA1-1(PR8) where the consensus glycosylationsequences had been removed (SEQ ID NO: 152).

The immunized mice were challenged on day 28 with an LD₉₀ (8×10³ EID) ofPR/8/34 virus administered intranasally. Mice were monitored daily for21 days following the challenge for survival, weight loss and clinicalpresentation. As shown in FIGS. 13A, 13B, 13C, PBS-immunized mice showedsigns of infection (weight loss and lower clinical scores) as early asthree days after challenge and died by day 21, while mice immunized with3.0 μg of recombinant STF2.HA1-2(PR8) (SEQ ID NO: 90) fusion proteindemonstrated enhanced protection with little to no observable clinicalsigns of infection or weight loss. Although animals immunized withSTF2.HA1-1 fusion proteins demonstrated significantly higher efficacythan animals immunized with PBS alone, these animals demonstrated lowerefficacy in terms of survival, clinical scores and overall weight loss,compared to animals receiving STF2.HA1-2(PR8) (SEQ ID NO: 90). Theseresults demonstrate that HA subunits linked to STF2 in Baculovirus areimmunogenic and efficacious, and suggest that STF2.HA1-1 (SEQ ID NO: 89)expressed in E. coli or Baculovirus is less immunogenic and efficaciousthan STF2.HA1-2 expressed in E. coli(SEQ ID NO: 90) or Baculovirus.

Example 10 Purification of STF2.HA1-2 (IND) (SEQ ID NO: 159) Materialsand Methods

Cell Banking:

E. coli cells engineered to express STF2.HA1-2(IND) (SEQ ID NO: 159)were adapted to culture in proprietary MRSF media, banked as glycerolstocks in 1 mL aliquots, and stored at −70° C.

MRSF media, pH 7.0 Composition g/L Glucose 10 KH₂PO₄ 7.8 (NH₄)₂SO4 2.33Citric Acid 1.0 MgSO₄(7H₂0) 1.0 CaCl₂ 0.04 Trace Metals 1 ml ThiamineHCl 0.01 Kanamycin 0.0075

Trace Metal Solution 1000x Component g/L EDTA, disodium 5 FeSO₄(7H₂O) 10ZnSO₄(7H₂O) 2 MnSO₄(H₂O) 2 CoCl₂(6H₂O) 0.2 CuSO₄(5H₂O) 0.1 Na₂MoO₄(2H₂O)0.2 H₃BO₃ 0.1 H₂O 1000 ml

Cell Scale-Up:

Two vials (2 ml, banked in MRSF media) of E. coli cells engineered toexpress STF2.HA1-2 (IND) (SEQ ID NO: 159) were retrieved from—−70° C.and inoculated into 500 ml of MRSF medium. The cells were expanded byincubation at 37° C. for 11.5 hours, at which time the OD₆₀₀ was 3.2.The cell scale up provided biomass to inoculate the productionbioreactor used in the fermentation.

Fermentation:

A 250 mL aliquot of scaled up cells was used to inoculate a 12 L workingvolume bioreactor containing 10 L of proprietary MRBR media. The culturewas incubated with constant stirring while maintaining pH at 7.0±0.1,temperature at 30±0.1° C., and dissolved oxygen (DO) at no less than30%, and was run in batch mode until the glucose was exhausted. Glucoseexhaustion is indicated by a sudden decrease in O₂ demand, and confirmedby measurement of glucose concentration on a YSI 2700 Select glucosemeter. The OD₆₀₀ for the culture at this time was 20.1 AU.

Thirty minutes after glucose exhaustion, proprietary feed media waspumped into the bioreactor at a controlled rate until 2 L of feed wasadded. The incubation continued for 5.5 hours under glucose-limitingconditions. At an OD₆₀₀ of 34 AU, protein expression in the culture wasinduced by the addition of IPTG to a final concentration of 2 mM andincubation with constant stirring for 2 hours. At the conclusion of theinduction period, cells were harvested by centrifugation in an AvantiJP-20 XP centrifuge for 20 minutes at 10,000 g. The final OD₆₀₀ for theculture was 58 AU and the total protein concentration as determined byBCA was 6.73 mg/mL. 1.16 kg of cell paste was recovered from 10.8 Lharvest and frozen at −20° C. The total bioreactor run time was 17hours. STF2.HA1-2 (IND) (SEQ ID NO: 159) production was confirmed by SDSPAGE.

MRBR Media, pH 7.0 Composition g/L Glucose 20 KH₂PO₄ 2.2 (NH₄)₂SO₄ 4.5Citric Acid 1.0 MgSO₄(7H₂0) 1.0 CaCl₂ 0.04 Trace Metals 1 ml ThiamineHCl 0.01 Antifoam 0.05 Kanamycin 0.0075

Feed media, pH 6.0 - additions to MRBR media (2 L final volume)Composition g/L Glucose 160 KH₂PO₄ 5.6 DL-Alanine 50.0 Citric Acid 3.0MgSO₄(7H₂0) 1.0 (added as 78 g/L solution) CaCl₂ 2.5 (added as 80 g/Lsolution) Trace Metals 24 ml FeSO₄ 7H₂O 0.75

Cell Disruption and Clarification:

The cell paste was thawed and resuspended into a 15% suspension of 50 mMTris, 25 mM NaCl, pH 8. The slurry was homogenized in an APV1000homogenizer three times at 12,000 psi. The lysate was adjusted to pH 4.0by the addition of acetic acid and centrifuged to separate soluble frominsoluble material. STF2.HA1-2 (IND) (SEQ ID NO: 159) along with mostother proteins partition to the insoluble fraction. The soluble materialwas discarded and the insoluble material (pellet) was dissolved in twotimes the initial lysate volume of 50 mM acetate, 1% TritonX-100, 8 MUrea, pH 4. After mixing in the homogenizer at 0 psi, the sample wascentrifuged and the supernatant was collected, filtered, and stored at4° C. The presence of STF2.HA1-2 (IND) (SEQ ID NO: 159) in the properphases (precipitate or supernatant) was confirmed by SDS PAGE.

Cation Exchange Chromatography (CEX):

The clarified material was applied to a 50 ml Poros 50HS column. Thisstep was designed to reduce the endotoxin concentration, reduce thenucleic acid concentration and increase the STF2.HA1-2 (IND) (SEQ ID NO:159) purity of the eluate as compare to the load. After loading, thecolumn was washed with five column volumes of 50 mM acetate, 1% TritonX-100, 8 M Urea, pH 4 followed by 10 column volumes of 50 mM acetate, 8M Urea, pH 4. The protein was eluted in a 0-100% gradient to 1 M NaCl.The target protein eluted at approximately 300 mM NaCl. The major peak(eluate) was pooled and stored at 4° C. The presence of STF2.HA1-2 (IND)(SEQ ID NO: 159) in the elution was confirmed by SDS PAGE. STF2.HA1-2(IND) (SEQ ID NO: 159) purification across this step was determined bycomparing the number and intensity of protein bands in the load materialto those in the elution.

Endotoxin Removal in Cation Exchange Chromatography (CEX):

Endotoxin is removed from eluting STF2.HA1-2 (IND) (SEQ ID NO: 159) withthe 50 mM acetate, 1% Triton X-100, 8 M Urea, pH 4 wash and with properelution conditions. This was tested using STF2.HA1-2 (VN) (SEQ ID NO:95) Comparing two separate cation exchange runs (Toso SP650M resin) withand without the 50 mM acetate, 1% Triton X-100, 8 M Urea, pH 4 wash (ERwash vs. no ER wash), the endotoxin concentration in the ER wash elutionat 150 mM NaCl shows significantly less endotoxin concentration that the250 mm NaCl elution from the same run, or in either of the elutionswithout the 50 mM acetate, 1% Triton X-100, 8 M Urea, pH 4 wash.

[protein] Elution [Endotoxin] [Endotoxin] by BCA UV Run (mM NaCl)(EU/mL) (EU/mg) (g/L) 280/260 Load N/A 4.4 × 10⁶ 1.8 × 10⁶ 2.39 — No ER100 7.9 × 10⁶ 4.2 × 10⁶ 1.44 1.05 wash 250 8.0 × 10⁶ 4.1 × 10⁶ 1.12 2.76ER 150  5,112 2,888 1.77 0.982 Wash 250 24,440 N/A N/A 0.545

Refolding:

The CEX eluate was dialyzed against 50 mM Tris, 8 M Urea, pH 8 buffer,and refolded by rapid dilution into 10 volumes of 50 mM Tris, pH 8.

Anion Exchange Chromatography (AEX):

The refolded protein was applied to a 25 ml GE Source Q column. Thisstep is designed to separate properly folded STF2.HA1-2 (IND) (SEQ IDNO: 159) from aggregates and host cell proteins and to reduce theendotoxin concentration of the eluate as compared to the load. Afterloading, the column was washed with five column volumes of 50 mM Tris,25 mM NaCl, pH 8. The protein was eluted in a 0-100% gradient to 1 MNaCl. A peak enriched for properly folded STF2.HA1-2(IND) (SEQ ID NO:159) with low endotoxin (1.7 EU/mL) eluted at approximately 150 mM NaCl.A second peak eluting at approximately 170 to 270 mM NaCl contains otherproteins including STF2.HA1-2 (IND) (SEQ ID NO: 159) aggregates.Fractions in first peak were pooled at stored at 4° C. Fractions fromthe first peak were kept for further processing. The presence ofSTF2.HA1-2 (IND) (SEQ ID NO: 159) in the first and second elutions wasconfirmed by SDS PAGE. STF2.HA1-2 (IND) (SEQ ID NO: 159) purificationacross this step was determined by comparing the number and intensity ofprotein bands in the load material to those in the elution.

Preparative Size Exclusion Chromatography (SEC):

The AEX eluate was run on a 320 ml SEC column in 2×5 mL fractionsagainst TBS. This step is designed to separate correctly foldedSTF2.HA1-2 (IND) (SEQ ID NO: 159) from other proteins. Each run resultedin elution of a single major peak which was collected, pooled, andfiltered through a 0.22 μm filter. The product was stored at −70° C.

The presence of STF2.HA1-2 (IND) (SEQ ID NO: 159) in the elution wasconfirmed by SDS PAGE. STF2.HA1-2 (IND) (SEQ ID NO: 159) purificationacross this step was determined by comparing the number and intensity ofprotein bands in the load material to those in the elution. The finalmaterial profile on SDS PAGE is a strong single band.

Results and Discussion

Final Characterization of Protein:

Recombinant STF2.HA1-2(IND) (SEQ ID NO: 159) protein was expressed in E.coli and purified to homogeneity. The final product was formulated in1×TBS (27.7 mM Tris, 2.7 mM KCl, 137 mM NaCl, pH 7.4; made from 10×stock, Teknova catalog #T9530) and stored at −70° C. in aliquots of 1.0ml. The final yield was 15.75 ml (45 ml at a concentration of 0.35mg/ml), with an endotoxin level of 1.1 EU/mg protein as determined bythe LAL method. The protein retained TLR5 biological activity asmeasured in a cell-based assay of cytokine release

SDS-PAGE:

Protein identity was determined, and purity estimated, by SDS-PAGE. Analiquot sample was diluted in SDS-PAGE sample buffer and diluent. Thesamples were boiled for 5 minutes and loaded onto a 4 to 12% SDSpolyacrylamide gel (Invitrogen NuPage) and electrophoresed. The gel wasstained with Coomassie R-250 to visualize protein bands. Positiveresults for STF2.HA1-2 (IND) (SEQ ID NO: 159) were indicated by a bandat about 75 kDa as compared to BioRad precision plus standards run onthe same gel. Purity is estimated by visually comparing the intensity ofthe STF2.HA1-2 (IND) (SEQ ID NO: 159) band to all other bands.

TLR5 Bioactivity Assay:

HEK293 cells constitutively express TLR5, and secrete several solublefactors, including IL-8, in response to TLR5 signaling. Cells wereseeded in 96-well microplates (50,000 cells/well), and test proteinswere added and incubated overnight. The next day, the conditioned mediumwas harvested, transferred to a clean 96-well microplate, and frozen at−20° C. After thawing, the conditioned medium was assayed for thepresence of IL-8 in a sandwich ELISA using an anti-human IL-8 matchedantibody pair (Pierce, #M801E and #M802B) following the manufacturer'sinstructions. Optical density was measured using a microplatespectrophotometer (FARCyte, Amersham).

Endotoxin Assay:

Endotoxin levels were determined using the QCL-1000 QuantitativeChromogenic LAL test kit (Cambrex), following the manufacturer'sinstructions for the microplate method.

Protein Assay:

Total protein concentration was determined using the Micro BCA(bicinchoninic acid) Assay (Pierce) in the microplate format, usingbovine serum albumin as a standard, according to the manufacturer'sinstructions.

UV280/260 Assay:

This assay is a measure of the nucleic acid concentration in a sample ascompared to the protein concentration. The absorbance maximum occurs atapproximately 260 nm for nucleic acids, while the absorbance maximumoccurs for proteins at approximately 280 nm. A higher ratio between theabsorbance at 280 nm versus the absorbance at 260 nm indicates a sampleenriched in protein over nucleic acids.

Example 11 Cloning, Expression, and Biochemical Characterization ofRecombinant Flagellin-Hemagglutinin Fusion Proteins Produced inDrosophila Materials and Methods

Cloning and Expression of HA in Drosophila:

RNA was isolated from the influenza A strain A/Puerto Rico/8/34 usingQIAamp MinElute Virus Spin Kit (Qiagen, Cat#57704) following themanufacturer's instructions. The HA RNA was reverse transcribed usingSuperScript III First Strand Synthesis System for RT-PCR (Invitrogen;Cat #18080-051) or SuperScript III One-Step RT-PCR System withPlatinum-Taq High Fidelity (Invitrogen; Cat#12574-030) following themanufacturer's instructions, and the cDNA was cloned using the TOPOcloning vector PCRII Zero Blunt (Invitrogen). The HA0s fragment (HA genewithout the signal sequence and trans-membrane domain) was thensub-cloned into pMT/STF2Δ at both the C-terminus and NH2 terminus of thecassette using PCR-based strategy to generate the constructs STF2Δ.HA0s(PR8) (SEQ ID NO: 160) and HA0s(PR8).STF2Δ (SEQ ID NO: 167)respectively. The HA gene was also cloned into the pMT/Bip/V5-his vectorwithout STF2Δ to generate the construct HA0s.his (SEQ ID NO: 170). Table10 list the constructs and the primers used in their construction.

TABLE 10 PR8 HA constructs for expression in Drosophila SEQ FOR REV DNAID Primer Primer Template NO: Construct SEQ ID NO: SEQ ID NO: SEQ ID NO:160 STF2Δ.HA0sHis 161 162 163 164 STF2Δ.HA0s 165 166 163 167 HA0s.STF2Δ168 169 163 170 HA0sHis 171 172 163 173 HA0s 174 175 163

Recombinant Protein Expression from Drosophila Dme1-2 Cells:

The HA0s.His (SEQ ID NO: 170) and STF2Δ.HA0s (SEQ ID NO: 164) plasmidswere co-transfected with the pCoBlast plasmid into Drosophila Dme1-2cells to generate Dme1-2 HA0sHis(PR8) and Dme1-2 STF2Δ.HA0s(PR8) stablecells. Dme1-2 stable cells were expanded from adherent to shakercultures in selection media [Drosophila Sfm medium (Invitrogen;Carlsbad, Calif.)+25 μg/ml blasticidin] and then expanded to a 12 Lproduction scale.

Recombinant protein expression was induced by the addition of CuSO₄ to afinal concentration of 0.5 mM. After incubation for 72 hours cells wereremoved from the conditioned medium by centrifugation. The conditionedmedium was then clarified by passing through a 0.22 μm filter and storedat 4° C.

Purification of HA0sHis(PR8) (SEQ ID NO: 176):

Conditioned medium was applied to a Chelating Sepharose column(GE/Amersham Biosciences; Piscataway, N.J.) charged with NiSO₄ andequilibrated in Buffer A [20 mM Tris, pH 8.0, 0.5 M NaCl] and eluted ina linear gradient with Buffer B [Buffer A+0.5 M imidazole]. The proteinwas further purified by fractionation on a Superdex 200 SEC column(GE/Amersham Biosciences; Piscataway, N.J.) equilibrated in 1×Tris-buffered saline (TBS), pH 8.0. HA0s.His₆(PR8) fractions werepooled, aliquoted and stored at −80° C.

SDS-PAGE and Western Blot Analysis:

Protein identity was determined, and purity estimated, by SDS-PAGE. Analiquot of 5 μg of each sample was diluted in SDS-PAGE sample bufferwith or without 100 mM DTT as a reductant. The samples were boiled for 5minutes and loaded onto a 10% SDS polyacrylamide gel (LifeGels; French'sForrest, New South Wales, AUS) and electrophoresed. The gel was stainedwith Coomassie R-250 (Bio-Rad; Hercules, Calif.) to visualize proteinbands. For western blot, 0.5 μg/lane total protein was electrophoresedas described above and the gels were then electro-transferred to a PVDFmembrane and blocked with 5% (w/v) dry milk before probing withanti-flagellin antibody (Inotek; Beverly, Mass.), anti-His₆.antibody(Invitrogen; Carlsbad, Calif.) or influenza A PR/8/34 convalescentimmune serum (described below under Protein Antigenicity ELISA). Afterprobing with alkaline phosphatase-conjugated secondary antibodies(Pierce; Rockford, Ill.), protein bands were visualized with an alkalinephosphatase chromogenic substrate (Promega; Madison, Wis.).

Protein Assay:

Total protein concentration was determined using the Micro BCA(bicinchoninic acid) Assay (Pierce; Rockford, Ill.) in the microplateformat, using bovine serum albumin as a standard, according to themanufacturer's instructions.

Endotoxin Assay:

Endotoxin levels were determined using the QCL-1000 QuantitativeChromogenic LAL test kit (Cambrex; E. Rutherford, N.J.), following themanufacturer's instructions for the microplate method.

TLR5 Bioactivity Assay:

HEK293 cells constitutively express TLR5, and secrete several solublefactors, including IL-8, in response to TLR5 signaling. Cells wereseeded in 96-well microplates (50,000 cells/well), and recombinantDrosophila conditioned medium containing either HA0sHis(PR8) (SEQ ID NO:176) or STF2Δ.HA0s(PR8) (SEQ ID NO: 177) was added. The next day, theconditioned medium was harvested, transferred to a clean 96-wellmicroplate, and frozen at −20° C. After thawing, the conditioned mediumwas assayed for the presence of IL-8 in a sandwich ELISA using ananti-human IL-8 matched antibody pair (Pierce; Rockford, Ill., #M801Eand #M802B) following the manufacturer's instructions. Optical densitywas measured using a microplate spectrophotometer (FARCyte, GE/Amersham;Piscataway, N.J.).

Protein Antigenicity ELISA:

Purified HA0s.His₆(PR8) (SEQ ID NO: 176) was tested by ELISA todetermine if the recombinant protein displayed correctly folded epitopesof HA. 96-well ELISA plates were coated overnight at 4° C. with serialdilutions in PBS (100 μl/well) of HA0s.His₆(PR8) (SEQ ID NO: 176)protein starting at 5 μg/ml. Plates were blocked with 200 μl/well ofAssay Diluent Buffer (ADB; BD Pharmingen) for one hour at roomtemperature, then washed three times in PBS-T. A fixed dose of primaryantibody was then added to each well. To assay HA reactivity, 100μl/well of a 1:10,000 dilution of non-immune or PR/8/34 convalescentimmune serum in ADB was added. PR/8/34 immune serum was generated inBALB/c mice (Jackson Laboratory, Bar Harbor, Me.) that received anexperimentally determined sublethal challenge dose of 8×10¹ egginfectious dosages (EID) of PR/8/34 influenza virus.

Animals were then allowed to convalesce for >21 days post-infection atwhich time immune serum was isolated and clarified. For ELISA of the 6×histidine tag, monoclonal antibody against 6× His (Invitrogen; Carlsbad,Calif.), or flagellin (Inotek; Beverly, Mass.) was added at 1 μg/ml inADB (100 μl/well) and the plates were incubated for 1 hr at roomtemperature or overnight at 4° C. The plates were then washed threetimes with PBS-T. HRP-labeled goat anti-mouse IgG antibodies (JacksonImmunochemical; West Grove, Pa.) diluted in ADB were added (100 μl/well)and the plates were incubated at room temperature for 1 hour. The plateswere washed three times with PBS-T. After adding TMB Ultra substrate(Pierce; Rockford, Ill.) and monitoring color development, A₄₅₀ wasmeasured on a microplate spectrophotometer (FARCyte, GE/Amersham;Piscataway, N.J.).

Results and Discussion

Characterization of Drosphila-Expressed HA0s Proteins:

Western blot analysis of conditioned media with anti-His₆ antibody(Invitrogen; Carlsbad, Calif.) confirmed expression of HA0sHis(PR8) (SEQID NO: 176) and western blot with anti-flagellin antibody (Inotek;Beverly, Mass.) confirmed expression of STF2Δ.HA0s(PR8) (SEQ ID NO: 177)by Drosophila Dme1-2 cells transfected with the corresponding expressionplasmid. Both proteins were recognized by western blot in non-reducedform with mouse PR/8/34 convalescent immune serum while reduction of theproteins with DTT abrogated recognition. This result indicates correctdisulfide bonding of the two secreted proteins. The STF2Δ.HA0s(PR8) (SEQID NO: 177) conditioned medium showed significant in vitro TLR5 activitywhile the HA0sHis₆(PR8) (SEQ ID NO: 176) medium did not, as expected.Finally, purified HA0sHis₆(PR8) (SEQ ID NO: 176) protein showedsignificant reactivity with influenza A PR8/34 convalescent immune serumby ELISA. These results indicate that both HA0s proteins are secretedfrom Drosophila Dme1-2 cells in a properly folded form.

Example 12 Immunogenicity and Efficacy of RecombinantFlagellin-Hemagglutinin Fusion Proteins Representing Viral Strain A/VietNam/1203/04 Materials and Methods

Animal Studies:

Female BALB/c mice were used at the age of 6-8 weeks. Mice were dividedinto groups of 15 and received subcutaneous (s.c) immunizations on days0 and 14 as follows:

TBS (phosphate buffered saline)

No immunization

10 μg of STF2.HA1-2(VN) (SEQ ID NO: 95) in TBS

3.0 μg of STF2.HA1-2(VN) (SEQ ID NO: 95) in PBS

1 μg of STF2.HA1-2(VN) (SEQ ID NO: 95) in TBS

Mice were bled on day 21 and sera were clarified by clotting andcentrifugation and stored at −20° C.

Serum Antibody Determination:

HA-specific IgG levels were determined by ELISA. 96-well ELISA plates(Costar (Cat #9018) Corning, N.Y.) were coated overnight at 4° C. with100 μl/well recombinant HA protein from A/Vietnam/1203/04, produced inBaculovirus, (BEIR catalog number NR-660) in PBS (1 μg/ml). Plates wereblocked with 300 μl/well of Assay Diluent Buffer (ADB; BD Pharmingen,(Cat#: 555213) (San Diego, Calif.) for two hours at 25° C. The plateswere washed three times in PBS+0.05% (v/v) Tween 20 (PBS-T). Dilutionsof the sera in ADB were added (100 μl/well) and the plates wereincubated for 1.5 hours at 25° C. The plates were washed three timeswith PBS-T. HRP-labeled goat anti-mouse IgG antibodies (JacksonImmunochemical, West Grove, Pa. (Cat#: 115-035-146)) diluted in ADB wereadded (100 μl/well) and the plates were incubated at 25° C. for 30minutes. The plates were washed three times with PBS-T. After adding TMBUltra substrate (Pierce (Cat 34028), Rockford, Ill.)) and monitoringcolor development, A₄₅₀ was measured on a SpectraMax 190 (MolecularDevices, Sunnyvale, Calif.) microplate spectrophotometer.

Influenza Virus Challenge of Mice:

To assess efficacy, mice were challenged on day 28 by intranasaladministration of 10 LD₉₀ (10× dose lethal to 90% of mice) (6×10³ EID)of influenza A/Viet Nam/1203/04. Animals were monitored daily for 21days following the challenge for survival and clinical presentation.

Preparation of Influenza H5N1 Stocks:

Influenza A/Vietnam/1203/04 (H5N1) was obtained from the Centers forDisease Control and Prevention (Atlanta, Ga.). Stocks of the virus wereprepared in 10-day-old embryonated chicken eggs, and aliquots of 0.5 mlwere stored at −80° C. until use.

Determination of Viral Titers:

TCID₅₀ (50% tissue culture infectious dose) was determined utilizingMDCK cells seeded into the wells of 96-well plate and grown toconfluency. A series of log₁₀ dilutions of the virus was inoculated ontothe plate in quadruplicates. The plate was then incubated for 48-72hours. The TCID₅₀ was determined by identifying the dilution at whichquadruplicate wells were ½ positive and ½ negative for viral growth. TheEID₅₀ was determined by inoculating log₁₀ dilutions of the stock into2-4 eggs per dilution. Eggs were incubated for 40-48 hours, and 1 ml ofthe allantoic fluid was harvested from each egg. EID₅₀ was calculated asthe dilution at which ½ of the eggs were negative and ½ of the eggs werepositive for infectious virus.

Results and Discussion

Induction of HA-Specific IgG Response Following Immunization withSTF2.HA1-2(VN) (SEQ ID NO: 95):

The immunogenicity of STF2.HA1-2(VN) (SEQ ID NO: 95) was examined byimmunizing BALB/c mice (15/group) subcutaneously on day 0 and 14 with adose range of 10, 3, and 1 μg protein. Negative control groups of micewere immunized with TBS or not immunized. HA-specific IgG responses wereexamined 7 days post boost (Day 21) by ELISA. The results demonstratethat immunization with 10, 3 or 1 μg of STF2.HA1-2(VN) (SEQ ID NO: 95)induced consistent and significant HA-specific IgG responses in adose-dependent manner (FIGS. 14-16).

Immunization with STF2.HA1-2(VN) (SEQ ID NO: 95) Provides Protectionfrom a Lethal Challenge with Influenza A:

The serological analysis described above demonstrated that immunizationof mice with STF2.HA1-2(VN) (SEQ ID NO: 95) generated an antibodyresponse that recognized native HA. In order to evaluate efficacy, thesame mice were challenged on day 28 with 10 LD₉₀ (6×10³EID) of A/VietNam/1203/04 virus administered intra-nasally. Mice were monitored dailyfor 21 days following the challenge for survival and clinicalpresentation. As shown in FIG. 17, TBS-immunized or naïve mice showedsigns of infection (weight loss and lower clinical scores) as early asfive days post-challenge and all mice died by day nine post-challenge.For the mice immunized with STF2.HA1-2(VN), there is a clearrelationship between the dose received and efficacy. Mice immunized with10 μg of STF2.HA1-2(VN) (SEQ ID NO: 95) demonstrated markedly enhancedprotection. Mice immunized with 3 or 1 μg of STF2.HA1-2(VN) (SEQ ID NO:95) demonstrated a modest to a negligible impact on efficacy,respectively. Clinical parameters were similarly related to dose andanimals receiving the highest dose of STF2.HA1-2(VN) (SEQ ID NO: 95)exhibited the mildest signs of disease. These results demonstrate thatE. coli-expressed STF2.HA1-2(VN) (SEQ ID NO: 95) induces HA-specificimmune responses that successfully protect BALB/c mice from a lethalchallenge with virulent influenza A virus in vivo.

A/Puerto Rico/8/34 HA SEQ ID NO: 1MKANLLVLLSALAAADADTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDSHNGKLCRLKGIAPLQLGKCNIAGWLLGNPECDPLLPVRSWSYIVETPNSENGICYPGDFIDYEELREQLSSVSSFERFEIFPKESSWPNHNTNGVTAACSHEGKSSFYRNLLWLTEKEGSYPKLKNSYVNKKGKEVLVLWGIHHPPNSKEQQNLYQNENAYVSVVTSNYNRRFTPEIAERPKVRDQAGRMNYYWTLLKPGDTIIFEANGNLIAPMYAFALSRGFGSGIITSNASMHECNTKCQTPLGAINSSLPYQNIHPVTIGECPKYVRSAKLRMVTGLRNIPSIQSRGLFGAIAGFIEGGWTGMIDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNTVIEKMNIQFTAVGKEFNKLEKRMENLNKKVDDGFLDIWTYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEIGNGCFEFYHKCDNECMESVRNGTYDYPKYSEESKLNREKVDGVKLESMGIYQILAIYSTVASSLVLLVSLGAISFWMCSNGSLQCRICIA/Viet Nam/1203/2004 HA SEQ ID NO: 2MEKIVLLFAIVSLVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKKHNGKLCDLDGVKPLILRDCSVAGWLLGNPMCDEFINVPEWSYIVEKANPVNDLCYPGDFNDYEELKHLLSRINHFEKIQIIPKSSWSSHEASLGVSSACPYQGKSSFFRNVVWLIKKNSTYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQTKLYQNPTTYISVGTSTLNQRLVPRIATRSKVNGQSGRMEFFWTILKPNDAINFESNGNFIAPEYAYKIVKKGDSTIMKSELEYGNCNTKCQTPMGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQRERRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHKCDNECMESVRNGTDYPQYSEEARLKREEISGVKLESIGIYQILSIYSTVASSLALAIMVAGLSLWMCSNGSLQCRA/Indonesia/5/2005 HA SEQ ID NO: 3MEKIVLLLAIVSLVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCDLDGVKPLILRDCSVAGWLLGNPMCDEFINVPEWSYIVEKANPTNDLCYPGSFNDYEELKHLLSRINHFEKIQIIPKSSWSDHEASSGVSSACPYLGSPSFFRNVVWLIKKNSTYPTIKKSYNNTNQEDLLVLWGIHHPNDAAEQTRLYQNPTTYISIGTSTLNQRLVPKIATRSKVNGQSGRMEFFWTILKPNDAINFESNGNFIAPEYAYKIVKKGDSAIMKSELEYGNCNTKCQTPMGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQRESRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHKCDNECMESIRNGTYNYPQYSEEARLKREEISGVKLESIGTYQILSIYSTVASSLALAIMMAGLSLWMCSNGSLQCRICIA/NewCaledonia/20/1999 SEQ ID NO: 4MKAKLLVLLCTFTATYADTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDSHNGKLCLLKGIAPLQLGNCSVAGWILGNPECELLISKESWSYIVETPNPENGTCYPGYFADYEELREQLSSVSSFERFEIFPKESSWPNHTVTGVSASCSHNGKSSFYRNLLWLTGKNGLYPNLSKSYVNNKEKEVLVLWGVHHPPNIGNQRALYHTENAYVSVVSSHYSRRFTPEIAKRPKVRDQEGRINYYWTLLEPGDTIIFEANGNLIAPWYAFALSRGFGSGIITSNAPMDECDAKCQTPQGAINSSLPFQNVHPVTIGECPKYVRSAKLRMVTGLRNIPSIQSRGLFGAIAGFIEGGWTGMVDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVIEKMNTQFTAVGKEFNKLERRMENLNKKVDDGFLDIWTYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEIGNGCFEFYHKCNNECMESVKNGTYDYPKYSEESKLNREKIDGVKLESMGVYQILAIYSTVASSLVLLVSLGAISFWMCSNGSLQCRICIA/South Carolina/1/18HA SEQ ID NO: 5MEARLLVLLCAFAATNADTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDSHNGKLCKLKGIAPLQLGKCNIAGWLLGNPECDLLLTASSWSYIVETSNSENGTCYPGDFIDYEELREQLSSVSSFEKFEIFPKTSSWPNHETTKGVTAACSYAGASSFYRNLLWLTKKGSSYPKLSKSYVNNKGKEVLVLWGVHHPPTGTDQQSLYQNADAYVSVGSSKYNRRFTPEIAARPKVRDQAGRMNYYWTLLEPGDTITFEATGNLIAPWYAFALNRGSGSGIITSDAPVHDCNTKCQTPHGAINSSLPFQNIHPVTIGECPKYVRSTKLRMATGLRNIPSIQSRGLFGAIAGFIEGGWTGMIDGWYGYHHQNEQGSGYAADQKSTQNAIDGITNKVNSVIEKMNTQFTAVGKEFNNLERRIENLNKKVDDGFLDIWTYNAELLVLLENERTLDFHDSNVRNLYEKVKSQLKNNAKEIGNGCFEFYHKCDDACMESVRNGTYDYPKYSEESKLNREEIDGVKLESMGVYQILAIYSTVASSLVLLVSLGAISFWMCSNGSLQCRICIA/Wisconsin/67/2005 HA SEQ ID NO: 6QKLPGNDNSTATLCLGHHAVPNGTIVKTITNDQIEVTNATELVQSSSTGGICDSPHQILDGENCTLIDALLGDPQCDGFQNKKWDLFVERSKAYSNCYPYDVPDYASLRSLVASSGTLEFNDESFNWTGVTQNGTSSACKRRSNNSFFSRLNWLTHLKFKYPALNVTMPNNEKFDKLYIWGVHHPGTDNDQIFLHAQASGRITVSTKRSQQTVIPNIGSRPRIRNIPSRISIYWTIVKPGDILLINSTGNLIAPRGYFKIRSGKSSIMRSDAPIGKCNSECITPNGSIPNDKPFQNVNRITYGACPRYVKQNTLKLATGMRNVPEKQTR A/X31 subtype H3N2 HA SEQ ID NO: 7QDLPGNDNSTATLCLGHHAVPNGTLVKTITDDQIEVTNATELVQSSSTGKICNNPHRILDGIDCTLIDALLGDPHCDVFQNETWDLFVERSKAFSNCYPYDVPDYASLRSLVASSGTLEFITEGFTWTGVIQNGGSNACKRGPGSGFFSRLNWLTKSGSTYPVLNVTMPNNDNFDKLYIWGIHHPSTNQEQTSLYVQASGRVTVSTRRSQQTIIPNIGSRPWVRGLSSRISIYWTIVKPGDVLVINSNGNLIAPRGYFKMRTGKSSIMRSDAPIDTCISECITPNGSIPNDKPFQNVNKITYGACPKYVKQNTLKLATGMRNVPEKQT PR/8 HA1-1 SEQ ID NO: 8SHNGKLCRLKGIAPLQLGKCNIAGWLLGNPECDPLLPVRSWSYIVETPNSENGICYPGDFIDYEELREQLSSVSSFERFEIFPKESSWPNHNTNGVTAACSHEGKSSFYRNLLWLTEKEGSYPKLKNSYVNKKGKEVLVLWGIHHPPNSKEQQNLYQNENAYVSVVTSNYNRRFTPEIAERPKVRDQAGRMNYYWTLLKPGDTIIFEANGNLIAPMYAFALSRGFGSGIITSNASMHECNTKCQTPLGAINSSLPYQNIHPVTIGECPKYVR PR/8 HA1-2 SEQ ID NO: 9KGIAPLQLGKCNIAGWLLGNPECDPLLPVRSWSYIVETPNSENGICYPGDFIDYEELREQLSSVSSFERFEIFPKESSWPNHNTNGVTAACSHEGKSSFYRNLLWLTEKEGSYPKLKNSYVNKKGKEVLVLWGIHHPPNSKEQQNLYQNENAYVSVVTSNYNRRFTPEIAERPKVRDQAGRMNYYWTLLKPGDTIIFEANGNLIAPMYAFALSRGFGSGIITS PR/8 HA1-3 SEQ ID NO: 10NSENGICYPGDFIDYEELREQLSSVSSFERFEIFPKESSWPNHNTNGVTAACSHEGKSSFYRNLLWLTEKEGSYPKLKNSYVNKKGKEVLVLWGIHHPPNSKEQQNLYQNENAYVSVVTSNYNRRFTPEIAERPKVRDQAGRMNYYWTLLKPGDTIIFEANGNLIAPMYAFALSRG VN HA1-1 SEQ ID NO: 11EKKHNGKLCDLDGVKPLILRDCSVAGWLLGNPMCDEFINVPEWSYIVEKANPVNDLCYPGDFNDYEELKHLLSRINHFEKIQIIPKSSWSSHEASLGVSSACPYQGKSSFFRNVVWLIKKNSTYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQTKLYQNPTTYISVGTSTLNQRLVPRIATRSKVNGQSGRMEFFWTILKPNDAINFESNGNFIAPEYAYKIVKKGDSTIMKSELEYGNCNTKCQTPMGAINSSMPFHNIHPLTIGECPKYVK VN HA1-2 SEQ ID NO: 12GVKPLILRDCSVAGWLLGNPMCDEFINVPEWSYIVEKANPVNDLCYPGDFNDYEELKHLLSRINHFEKIQIIPKSSWSSHEASLGVSSACPYQGKSSFFRNVVWLIKKNSTYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQTKLYQNPTTYISVGTSTLNQRLVPRIATRSKVNGQSGRMEFFWTILKPNDAINFESNGNFIAPEYAYKIVKKGDSTIMKSE VN HA1-3 SEQ ID NO: 13NDLCYPGDFNDYEELKHLLSRINHFEKIQIIPKSSWSSHEASLGVSSACPYQGKSSFFRNVVWLIKKNSTYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQTKLYQNPTTYISVGTSTLNQRLVPRIATRSKVNGQSGRMEFFWTILKPNDAINFESNGNFIAPEYAYKIVKKG IND HA1-1 SEQ ID NO: 14EKTHNGKLCDLDGVKPLILRDCSVAGWLLGNPMCDEFINVPEWSYIVEKANPTNDLCYPGSFNDYEELKHLLSRINHFEKIQIIPKSSWSDHEASSGVSSACPYLGSPSFFRNVVWLIKKNSTYPTIKKSYNNTNQEDLLVLWGIHHPNDAAEQTRLYQNPTTYISIGTSTLNQRLVPKIATRSKVNGQSGRMEFFWTILKPNDAINFESNGNFIAPEYAYKIVKKGDSAIMKSELEYGNCNTKCQTPMGAINSSMPFHNIHPLTIGECPKYVK IND HA1-2 SEQ ID NO: 15GVKPLILRDCSVAGWLLGNPMCDEFINVPEWSYIVEKANPTNDLCYPGSFNDYEELKHLLSRINHFEKIQIIPKSSWSDHEASSGVSSACPYLGSPSFFRNVVWLIKKNSTYPTIKKSYNNTNQEDLLVLWGIHHPNDAAEQTRLYQNPTTYISIGTSTLNQRLVPKIATRSKVNGQSGRMEFFWTILKPNDAINFESNGNFIAPEYAYKIVKKGDSAIMKSE IND HA1-3 SEQ ID NO: 16NDLCYPGSFNDYEELKHLLSRINHFEKIQIIPKSSWSDHEASSGVSSACPYLGSPSFFRNVVWLIKKNSTYPTIKKSYNNTNQEDLLVLWGIHHPNDAAEQTRLYQNPTTYISIGTSTLNQRLVPKIATRSKVNGQSGRMEFFWTILKPNDAINFESNGNFIAPEYAYKIVKKG NC HA1-1 SEQ ID NO: 17SHNGKLCLLKGIAPLQLGNCSVAGWILGNPECELLISKESWSYIVETPNPENGTCYPGYFADYEELREQLSSVSSFERFEIFPKESSWPNHTVTGVSASCSHNGKSSFYRNLLWLTGKNGLYPNLSKSYVNNKEKEVLVLWGVHHPPNIGNQRALYHTENAYVSVVSSHYSRRFTPEIAKRPKVRDQEGRINYYWTLLEPGDTIIFEANGNLIAPWYAFALSRGFGSGIITSNAPMDECDAKCQTPQGAINSSLPFQNVHPVTIGECPKYVR NC HA1-2 SEQ ID NO: 18KGIAPLQLGNCSVAGWILGNPECELLISKESWSYIVETPNPENGTCYPGYFADYEELREQLSSVSSFERFEIFPKESSWPNHTVTGVSASCSHNGKSSFYRNLLWLTGKNGLYPNLSKSYVNNKEKEVLVLWGVHHPPNIGNQRALYHTENAYVSVVSSHYSRRFTPEIAKRPKVRDQEGRINYYWTLLEPGDTIIFEANGNLIAPWYAFALSRGFGSGIITS NC HA1-3 SEQ ID NO: 19NPENGTCYPGYFADYEELREQLSSVSSFERFEIFPKESSWPNHTVTGVSASCSHNGKSSFYRNLLWLTGKNGLYPNLSKSYVNNKEKEVLVLWGVHHPPNIGNQRALYHTENAYVSVVSSHYSRRFTPEIAKRPKVRDQEGRINYYWTLLEPGDTIIFEANGNLIAPWYAFALSRG WIS HA1-1 SEQ ID NO: 20QSSSTGGICDSPHQILDGENCTLIDALLGDPQCDGFQNKKWDLFVERSKAYSNCYPYDVPDYASLRSLVASSGTLEFNDESFNWTGVTQNGTSSACKRRSNNSFFSRLNWLTHLKFKYPALNVTMPNNEKFDKLYIWGVHHPGTDNDQIFLHAQASGRITVSTKRSQQTVIPNIGSRPRIRNIPSRISIYWTIVKPGDILLINSTGNLIAPRGYFKIRSGKSSIMRSDAPIGKCNSECITPNGSIPNDKPFQNVNRITYGACPRYVK WIS HA1-2 SEQ ID NO: 21SPHQILDGENCTLIDALLGDPQCDGFQNKKWDLFVERSKAYSNCYPYDVPDYASLRSLVASSGTLEFNDESFNWTGVTQNGTSSACKRRSNNSFFSRLNWLTHLKFKYPALNVTMPNNEKFDKLYIWGVHHPGTDNDQIFLHAQASGRITVSTKRSQQTVIPNIGSRPRIRNIPSRISIYWTIVKPGDILLINSTGNLIAPRGYFKIRSGKSSIMRSD WIS HA1-3 SEQ ID NO: 22SNCYPYDVPDYASLRSLVASSGTLEFNDESFNWTGVTQNGTSSACKRRSNNSFFSRLNWLTHLKFKYPALNVTMPNNEKFDKLYIWGVHHPGTDNDQIFLHAQASGRITVSTKRSQQTVIPNIGSRPRIRNIPSRISIYWTIVKPGDILLINSTGNLIAPRGYFKIRSG A/Puerto Rico/8/34 HA0sSEQ ID NO: 23DTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDSHNGKLCRLKGIAPLQLGKCNIAGWLLGNPECDPLLPVRSWSYIVETPNSENGICYPGDFIDYEELREQLSSVSSFERFEIFPKESSWPNHNTNGVTAACSHEGKSSFYRNLLWLTEKEGSYPKLKNSYVNKKGKEVLVLWGIHHPPNSKEQQNLYQNENAYVSVVTSNYNRRFTPEIAERPKVRDQAGRMNYYWTLLKPGDTIIFEANGNLIAPMYAFALSRGFGSGIITSNASMHECNTKCQTPLGAINSSLPYQNIHPVTIGECPKYVRSAKLRMVTGLRNIPSIQSRGLFGAIAGFIEGGWTGMIDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNTVIEKMNIQFTAVGKEFNKLEKRMENLNKKVDDGFLDIWTYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEIGNGCFEFYHKCDNECMESVRNGTYDYPKYSEESKLNREKVDGVKLESMGIYQPR/8 HA1-2mut SEQ ID NO: 24KGAAPLQLGKCNIAGWLLGNPECDPLLPVRSWSDIAETPNSENGICYPGDFIDYEELREQLSSVSSFERFEIFPKESSWPNHNTNGVTAACSHEGKSSFYRNLLWLTEKEGSYPKLKNSYVNKKGKEVLVLWGIHHPPNSKEQQNLYQNENAYVSVVTSNYNRRFTPEIAERPKVRDQAGRMNYYWTLLKPGDTIIFEANGNLIAPMYAFALSRGFGSGIITS PR/8 HA1-3mut SEQ ID NO: 25NSENEICYPGDFIDKEELREQLSSVSSFERFEIFPKESSWPNHNTNGVTAACSHEGKSSFYRNLLWLTEKEGSYPKLKNSYVNKKGKEVLVLWGIHHPPNSKEQQNLYQNENAYVSVVTSNYNRRFTPEIAERPKVRDQAGRMNYYWTLLKPGDTIIFEANGNLIAPMYAAALSRG VN HA1-2mutSEQ ID NO: 26GAKPLSLRDCSVAGWLLGNPMCDEFINVPEWSDIAEKANPVNDLCYPGDFNDYEELKHLLSRINHFEKIQIIPKSSWSSHEASLGVSSACPYQGKSSFFRNVVWLIKKNSTYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQTKLYQNPTTYISVGTSTLNQRLVPRIATRSKVNGQSGRMEFFWTILKPNDAINFESNGNFIAPEYAYKIVKKGDSTIMKSE VN HA1-3mut SEQ ID NO: 27NDLCYPGDFNDKEELKHLLSRINHFEKIQIIPKSSWSSHEASLGVSSACPYQGKSSFFRNVVWLIKKNSTYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQTKLYQNPTTYISVGTSTLNQRLVPRIATRSKVNGQSGRMEFFWTILKPNDAINFESNGNFIAPEYAYKIAKKG IND HA1-2mutSEQ ID NO: 28GAKPLSLRDCSVAGWLLGNPMCDEFINVPEWSDIAEKANPTNDLCYPGSFNDYEELKHLLSRINHFEKIQIIPKSSWSDHEASSGVSSACPYLGSPSFFRNVVWLIKKNSTYPTIKKSYNNTNQEDLLVLWGIHHPNDAAEQTRLYQNPTTYISIGTSTLNQRLVPKIATRSKVNGQSGRMEFFWTILKPNDAINFESNGNFIAPEYAYKIVKKGDSAIMKSE IND HA1-3mut SEQ ID NO: 29NDLCYPGSFNDKEELKHLLSRINHFEKIQIIPKSSWSDHEASSGVSSACPYLGSPSFFRNVVWLIKKNSTYPTIKKSYNNTNQEDLLVLWGIHHPNDAAEQTRLYQNPTTYISIGTSTLNQRLVPKIATRSKVNGQSGRMEFFWTILKPNDAINFESNGNFIAPEYAYKIAKKG NC HA1-2mut SEQ ID NO: 30KGAAPLQLGNCSVAGWILGNPECELLISKESWSDIAETPNPENGTCYPGYFADYEELREQLSSVSSFERFEIFPKESSWPNHTVTGVSASCSHNGKSSFYRNLLWLTGKNGLYPNLSKSYVNNKEKEVLVLWGVHHPPNIGNQRALYHTENAYVSVVSSHYSRRFTPEIAKRPKVRDQEGRINYYWTLLEPGDTIIFEANGNLIAPWYAFALSRGFGSGIITS NC HA1-3mut SEQ ID NO: 31NPENETCYPGYFADKEELREQLSSVSSFERFEIFPKESSWPNHTVTGVSASCSHNGKSSFYRNLLWLTGKNGLYPNLSKSYVNNKEKEVLVLWGVHHPPNIGNQRALYHTENAYVSVVSSHYSRRFTPEIAKRPKVRDQEGRINYYWTLLEPGDTIIFEANGNLIAPWYAAALSRG WIS HA1-2mutSEQ ID NO: 32SPHQALDGENCTLIDALLGDPQCDGFQNKKWDDFAERSKAYSNCYPYDVPDYASLRSLVASSGTLEFNDESFNWTGVTQNGTSSACKRRSNNSFFSRLNWLTHLKFKYPALNVTMPNNEKFDKLYIWGVHHPGTDNDQIFLHAQASGRITVSTKRSQQTVIPNIGSRPRIRNIPSRISIYWTIVKPGDILLINSTGNLIAPRGYFKIRSGKSSIMRSD WIS HA1-3mut SEQ ID NO: 33SNCYPKDSPDEASLRSLVASSGTDEFNDESFNWTGVTQNGTSSACKRRSNNSFFSRLNWLTHLKFKYPALNVTMPNNEKFDKLYIWGVHHPGTDNDQIFLHAQASGRITVSTKRSQQTVIPNIGSRPRIRNIPSRISIYWTIVKPGDILLINSTGNLIAPRGYFKIRSGA/Swine/Hong Kong/9/98 HA (ACCESSION BAB85618) SEQ ID NO: 34DKICIGYQSTNSTETVDTLTETNVPVTHAKELLHTEHNGMLCATNLGHPLILDTCTIEGLIYGNPSCDLLLGGREWSYIVERPSAVNGMCYPGNVENLEELRSLFSSASSYQRIQIFPDTIWNVSYSGTSKACSDSFYRSMRWLTQKNNAYPIQDAQYTNNRGKSILFMWGINHPPTDTVQTNLYTRTDTTTSVTTEDINRTFKPVIGPRPLVNGLHGRIDYYWSVLKPGQTLRVRSNGNLIAPWYGHILSGESHGRILKTDLNSGNCVVQCQTERGGLNTTLPFHNVSKYAFGNCPKYVGVKSLKLAVGLRNVPARSSRA/Aichi/2/1968 HA (ACCESSION BAF37221) SEQ ID NO: 35MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITDDQIEVTNATELVQSSSTGKICNNPHRILDGIDCTLIDALLGDPHCDVFQNETWDLFVERSKAFSNCYPYDVPDYASLRSLVASSGTLEFITEGFTWTGVTQNGGSNACKRGPGSGFFSRLNWLTKSGSTYPVLNVTMPNNDNFDKLYIWGIHHPSTNQEQTSLYVQASGRVTVSTRRSQQTIIPNIGSRPWVRGLSSRISIYWTIVKPGDVLVINSNGNLIAPRGYFKMRTGKSSIMRSDAPIDTCISECITPNGSIPNDKPFQNVNKITYGACPKYVKQNTLKLATGMRNVPEKQTRGLFGAIAGFIENGWEGMIDGWYGFRHQNSEGTGQAADLKSTQAAIDQINGKLNRVIEKTNEKFHQIEKEFSEVEGRIQDLEKYVEDTKIDLWSYNAELLVALENQHTIDLTDSEMNKLFEKTRRQLRENAEEMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVELKSGYKDWILWISFAISCFLLCVVLLGFIMWACQRGNIRCNICIB/Lee/40 HA (Accession NP_056660) SEQ ID NO: 36MKAIIVLLMVVTSNADRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTPTKSHFANLKGTQTRGKLCPNCFNCTDLDVALGRPKCMGNTPSAKVSILHEVKPATSGCFPIMHDRTKIRQLPNLLRGYENIRLSTSNVINTETAPGGPYKVGTSGSCPNVANGNGFFNTMAWVIPKDNNKTAINPVTVEVPYICSEGEDQITVWGFHSDDKTQMERLYGDSNPQKFTSSANGVTTHYVSQIGGFPNQTEDEGLKQSGRIVVDYMVQKPGKTGTIVYQRGILLPQKVWCASGRSKVIKGSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLKERGFFGAIAGFLEGGWEGMIAGWHGYTSHGAHGVAVAADLKSTQEAINKITKNLNYLSELEVKNLQRLSGAMNELHDEILELDEKVDDLRADTISSQIELAVLLSNEGIINSEDEHLLALERKLKKMLGPSAVEIGNGCFETKHKCNQTCLDRIAAGTFNAGDFSLPTFDSLNITAASLNDDGLDNHTILLYYSTAASSLAVTLMIAIFIVYMVSRDNVSCSICL C/Johannesburg/1/66 (ACCESSION AAW73083) SEQ ID NO: 37MFFSLLLVLGLTEAEKIKICLQKQVNSSFSLHNGFGGNLYATEEKRMFELVKPKAGASVLNQSTWIGFGDSRTDKSNSAFPRSADVSAKTADKFRSLSGGSLMLSMFGPPGKVDYLYQGCGKHKVFYEGVNWSPHAAINCYRKNWTDIKLNFQKNIYELASQSHCMSLVNALDKTIPLQVTAGTAGNCNNSFLKNPALYTQEVKPSENKCGKENLAFFTLPTQFGTYECKLHLVASCYFIYDSKEVYNKRGCDNYFQVIYDSSGKVVGGLDNRVSPYTGNSGDTPTMQCDMLQLKPGRYSVRSSPRFLLMPERSYCFDMKEKGPVTAVQSIWGKGRESDYAVDQACLSTPGCMLIQKQKPYIGEADDHHGDQEMRELLSGLDYEARCISQSGWVNETSPFTEKYLLPPKFGRCPLAAKEESIPKIPDGLLIPTSGTDTTVTKPKSRIFGIDDLIIGLLFVAIVEAGIGGYLLGSRKESGGGVTKESAEKGFEKIGNDIQILKSSINIAIEKLNDRISHDEQAIRDLTLEIENARSEALLGELGIIRALLVGNISIGLQESLWELASEITNRAGDLAVEVSPGCWIIDNNICDQSCQNFIFKFNETAPVPTIPPLDTKIDLQSDPFYWGSSLGLAITATISLAALVISGIAICRTK B/Lee/40 HA1-1 SEQ ID NO: 38TTTPTKSHFANLKGTQTRGKLCPNCFNCTDLDVALGRPKCMGNTPSAKVSILHEVKPATSGCFPIMHDRTKIRQLPNLLRGYENIRLSTSNVINTETAPGGPYKVGTSGSCPNVANGNGFFNTMAWVIPKDNNKTAINPVTVEVPYICSEGEDQITVWGFHSDDKTQMERLYGDSNPQKFTSSANGVTTHYVSQIGGFPNQTEDEGLKQSGRIVVDYMVQKPGKTGTIVYQRGILLPQKVWCASGRSKVIKGSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVK B/Lee/40 HA1-2 SEQ ID NO: 39KGTQTRGKLCPNCFNCTDLDVALGRPKCMGNTPSAKVSILHEVKPATSGCFPIMHDRTKIRQLPNLLRGYENIRLSTSNVINTETAPGGPYKVGTSGSCPNVANGNGFFNTMAWVIPKDNNKTAINPVTVEVPYICSEGEDQITVWGFHSDDKTQMERLYGDSNPQKFTSSANGVTTHYVSQIGGFPNQTEDEGLKQSGRIVVDYMVQKPGKTGTIVYQRGILLPQKVWCASGRSKVIKG A/Aichi/2/68 HA1-1SEQ ID NO: 40QSSSTGKICNNPHRILDGIDCTLIDALLGDPHCDVFQNETWDLFVERSKAFSNCYPYDVPDYASLRSLVASSGTLEFITEGFTWTGVTQNGGSNACKRGPGSGFFSRLNWLTKSGSTYPVLNVTMPNNDNFDKLYIWGIHHPSTNQEQTSLYVQASGRVTVSTRRSQQTIIPNIGSRPWVRGLSSRISIYWTIVKPGDVLVINSNGNLIAPRGYFKMRTGKSSIMRSDAPIDTCISECITPNGSIPNDKPFQNVNKITYGACPKYVK A/Aichi/2/68 HA1-2 SEQ ID NO: 41HRILDGIDCTLIDALLGDPHCDVFQNETWDLFVERSKAFSNCYPYDVPDYASLRSLVASSGTLEFITEGFTWTGVTQNGGSNACKRGPGSGFFSRLNWLTKSGSTYPVLNVTMPNNDNFDKLYIWGIHHPSTNQEQTSLYVQASGRVTVSTRRSQQTIIPNIGSRPWVRGLSSRISIYWTIVKPGDVLVINSNGNLIAPRGYFKMRTGKSSIMRSD B/Malaysia/2506/2004 HA (Accession ISDN126672)SEQ ID NO: 42IVLLMVVTSNADRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTPTKSHFANLKGTETRGKLCPKCLNCTDLDVALGRPKCTGNIPSARVSILHEVRPVTSGCFPIMHDRTKIRQLPNLLRGYEHIRLSTHNVINAENAPGGSYKIGTSGSCPNVTNGNGFFATMAWAVPKNDNNKTATNSLTIEVPYICTEGEDQITVWGFHSDNEAQMAKLYGDSKPQKFTSSANGVTTHYVSQIGGFPNQTEDGGLPQSGRIVVDYMVQKSGKTGTITYQRGILLPQKVWCASGRSKVIKGSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLKERB/Ohio/1/2005 HA (Accession ISDN133312) SEQ ID NO: 43DRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTPTKSHFANLKGTKTRGKLCPKCLNCTDLDVALGRPKCTGNIPSAEVSILHEVRPVTSGCFPIMHDRTKIRQLPNLLRGYEHIRLSTHNVINAEKAPGGPYKIGTSGSCPNVTNGNGFFATMAWAVPKNDNNKTATNSLTIEVPYICTEGEDQITIWGFHSDSETQMAKLYGDSKPQKFTSSANGVTTHYVSQIGGFPNQTEDGGLPQSGRIVVDYMVQKSGKTGTITYQRGILLPQKVWCASGRSKVIKGSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLKERGFB/Shanghai/361/2002 HA (Accession ISDN80784) SEQ ID NO: 44DRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTPIKSHFANLKGTRTRGKLCPDCLNCTDLDVALGRPMCVGTTPSAKASILHEVRPVTSGCFPIMHDRTKIRQLPNLLRGYENIRLSTQNVIDAEKALGGPYRLGTSGSCPNATSKSGFFATMAWAVPKDNNKNATNPLTVEVPYICTEGEDQITVWGFHSDDKTQMKNLYGDSNPQKFTSSANGVTTHYVSQIGGFPDQTEDGGLPQSGRIVVDYMVQKPGKTGTIVYQRGVLLPQKVWCASGRSKVIKGSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGHCPIWVKTPLKLANGTKYRP B/Malaysia/2506/2004 HA1-1 SEQ ID NO: 45TTTPTKSHFANLKGTETRGKLCPKCLNCTDLDVALGRPKCTGNIPSARVSILHEVRPVTSGCFPIMHDRTKIRQLPNLLRGYEHIRLSTHNVINAENAPGGSYKIGTSGSCPNVTNGNGFFATMAWAVPKNDNNKTATNSLTIEVPYICTEGEDQITVWGFHSDNEAQMAKLYGDSKPQKFTSSANGVTTHYVSQIGGFPNQTEDGGLPQSGRIVVDYMVQKSGKTGTITYQRGILLPQKVWCASGRSKVIKGSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVK B/Malaysia/2506/2004 HA1-2SEQ ID NO: 46KGTETRGKLCPKCLNCTDLDVALGRPKCTGNIPSARVSILHEVRPVTSGCFPIMHDRTKIRQLPNLLRGYEHIRLSTHNVINAENAPGGSYKIGTSGSCPNVTNGNGFFATMAWAVPKNDNNKTATNSLTIEVPYICTEGEDQITVWGFHSDNEAQMAKLYGDSKPQKFTSSANGVTTHYVSQIGGFPNQTEDGGLPQSGRIVVDYMVQKSGKTGTITYQRGILLPQKVWCASGRSKVIKG B/Ohio/1/2005 HA1-1SEQ ID NO: 47TTTPTKSHFANLKGTKTRGKLCPKCLNCTDLDVALGRPKCTGNIPSAEVSILHEVRPVTSGCFPIMHDRTKIRQLPNLLRGYEHIRLSTHNVINAEKAPGGPYKIGTSGSCPNVTNGNGFFATMAWAVPKNDNNKTATNSLTIEVPYICTEGEDQITIWGFHSDSETQMAKLYGDSKPQKFTSSANGVTTHYVSQIGGFPNQTEDGGLPQSGRIVVDYMVQKSGKTGTITYQRGILLPQKVWCASGRSKVIKGSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVK B/Ohio/1/2005 HA1-2 SEQ ID NO: 48KGTKTRGKLCPKCLNCTDLDVALGRPKCTGNIPSAEVSILHEVRPVTSGCFPIMHDRTKIRQLPNLLRGYEHIRLSTHNVINAEKAPGGPYKIGTSGSCPNVTNGNGFFATMAWAVPKNDNNKTATNSLTIEVPYICTEGEDQITIWGFHSDSETQMAKLYGDSKPQKFTSSANGVTTHYVSQIGGFPNQTEDGGLPQSGRIVVDYMVQKSGKTGTITYQRGILLPQKVWCASGRSKVIKGB/Shanghai/361/2002 HA1-1 SEQ ID NO: 49TTTPIKSHFANLKGTRTRGKLCPDCLNCTDLDVALGRPMCVGTTPSAKASILHEVRPVTSGCFPIMHDRTKIRQLPNLLRGYENIRLSTQNVIDAEKALGGPYRLGTSGSCPNATSKSGFFATMAWAVPKDNNKNATNPLTVEVPYICTEGEDQITVWGFHSDDKTQMKNLYGDSNPQKFTSSANGVTTHYVSQIGGFPDQTEDGGLPQSGRIVVDYMVQKPGKTGTIVYQRGVLLPQKVWCASGRSKVIKGSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGHCPIWVK B/Shanghai/361/2002 HA1-2SEQ ID NO: 50KGTRTRGKLCPDCLNCTDLDVALGRPMCVGTTPSAKASILHEVRPVTSGCFPIMHDRTKIRQLPNLLRGYENIRLSTQNVIDAEKALGGPYRLGTSGSCPNATSKSGFFATMAWAVPKDNNKNATNPLTVEVPYICTEGEDQITVWGFHSDDKTQMKNLYGDSNPQKFTSSANGVTTHYVSQIGGFPDQTEDGGLPQSGRIVVDYMVQKPGKTGTIVYQRGVLLPQKVWCASGRSKVIKG STF2.blp SEQ ID NO: 51ATGGCACAAGTAATCAACACTAACAGTCTGTCGCTGCTGACCCAGAATAACCTGAACAAATCCCAGTCCGCACTGGGCACCGCTATCGAGCGTCTGTCTTCTGGTCTGCGTATCAACAGCGCGAAAGACGATGCGGCAGGTCAGGCGATTGCTAACCGTTTCACCGCGAACATCAAAGGTCTGACTCAGGCTTCCCGTAACGCTAACGACGGTATCTCCATTGCGCAGACCACTGAAGGCGCGCTGAACGAAATCAACAACAACCTGCAGCGTGTGCGTGAACTGGCGGTTCAGTCTGCTAACAGCACCAACTCCCAGTCTGACCTCGACTCCATCCAGGCTGAAATCACCCAGCGCCTGAACGAAATCGACCGTGTATCCGGCCAGACTCAGTTCAACGGCGTGAAAGTCCTGGCGCAGGACAACACCCTGACCATCCAGGTTGGCGCCAACGACGGTGAAACTATCGATATCGATCTGAAGCAGATCAACTCTCAGACCCTGGGTCTGGACTCACTGAACGTGCAGAAAGCGTATGATGTGAAAGATACAGCAGTAACAACGAAAGCTTATGCCAATAATGGTACTACACTGGATGTATCGGGTCTTGATGATGCAGCTATTAAAGCGGCTACGGGTGGTACGAATGGTACGGCTTCTGTAACCGGTGGTGCGGTTAAATTTGACGCAGATAATAACAAGTACTTTGTTACTATTGGTGGCTTTACTGGTGCTGATGCCGCCAAAAATGGCGATTATGAAGTTAACGTTGCTACTGACGGTACAGTAACCCTTGCGGCTGGCGCAACTAAAACCACAATGCCTGCTGGTGCGACAACTAAAACAGAAGTACAGGAGTTAAAAGATACACCGGCAGTTGTTTCAGCAGATGCTAAAAATGCCTTAATTGCTGGCGGCGTTGACGCTACCGATGCTAATGGCGCTGAGTTGGTCAAAATGTCTTATACCGATAAAAATGGTAAGACAATTGAAGGCGGTTATGCGCTTAAAGCTGGCGATAAGTATTACGCCGCAGATTACGATGAAGCGACAGGAGCAATTAAAGCTAAAACTACAAGTTATACTGCTGCTGACGGCACTACCAAAACAGCGGCTAACCAACTGGGTGGCGTAGACGGTAAAACCGAAGTCGTTACTATCGACGGTAAAACCTACAATGCCAGCAAAGCCGCTGGTCATGATTTCAAAGCACAACCAGAGCTGGCGGAAGCAGCCGCTAAAACCACCGAAAACCCGCTGCAGAAAATTGATGCCGCGCTGGCGCAGGTGGATGCGCTGCGCTCTGATCTGGGTGCGGTACAAAACCGTTTCAACTCTGCTATCACCAACCTGGGCAATACCGTAAACAATCTGTCTGAAGCGCGTAGCCGTATCGAAGATTCCGACTACGCGACCGAAGTTTCCAACATGTCTCGCGCGCAGATTCTGCAGCAGGCCGGTACTTCCGTTCTGGCGCAGGCTAACCAGGTCCCGCAGAACGTGCTGAGCCTGTTACGT STF2.SG SEQ ID NO: 52ATGGCACAAGTAATCAACACTAACAGTCTGTCGCTGCTGACCCAGAATAACCTGAACAAATCCCAGTCCGCACTGGGCACCGCTATCGAGCGTCTGTCTTCTGGTCTGCGTATCAACAGCGCGAAAGACGATGCGGCAGGTCAGGCGATTGCTAACCGTTTCACCGCGAACATCAAAGGTCTGACTCAGGCTTCCCGTAACGCTAACGACGGTATCTCCATTGCGCAGACCACTGAAGGCGCGCTGAACGAAATCAACAACAACCTGCAGCGTGTGCGTGAACTGGCGGTTCAGTCTGCTAACAGCACCAACTCCCAGTCTGACCTCGACTCCATCCAGGCTGAAATCACCCAGCGCCTGAACGAAATCGACCGTGTATCCGGCCAGACTCAGTTCAACGGCGTGAAAGTCCTGGCGCAGGACAACACCCTGACCATCCAGGTTGGCGCCAACGACGGTGAAACTATCGATATCGATCTGAAGCAGATCAACTCTCAGACCCTGGGTCTGGACTCACTGAACGTGCAGAAAGCGTATGATGTGAAAGATACAGCAGTAACAACGAAAGCTTATGCCAATAATGGTACTACACTGGATGTATCGGGTCTTGATGATGCAGCTATTAAAGCGGCTACGGGTGGTACGAATGGTACGGCTTCTGTAACCGGTGGTGCGGTTAAATTTGACGCAGATAATAACAAGTACTTTGTTACTATTGGTGGCTTTACTGGTGCTGATGCCGCCAAAAATGGCGATTATGAAGTTAACGTTGCTACTGACGGTACAGTAACCCTTGCGGCTGGCGCAACTAAAACCACAATGCCTGCTGGTGCGACAACTAAAACAGAAGTACAGGAGTTAAAAGATACACCGGCAGTTGTTTCAGCAGATGCTAAAAATGCCTTAATTGCTGGCGGCGTTGACGCTACCGATGCTAATGGCGCTGAGTTGGTCAAAATGTCTTATACCGATAAAAATGGTAAGACAATTGAAGGCGGTTATGCGCTTAAAGCTGGCGATAAGTATTACGCCGCAGATTACGATGAAGCGACAGGAGCAATTAAAGCTAAAACTACAAGTTATACTGCTGCTGACGGCACTACCAAAACAGCGGCTAACCAACTGGGTGGCGTAGACGGTAAAACCGAAGTCGTTACTATCGACGGTAAAACCTACAATGCCAGCAAAGCCGCTGGTCATGATTTCAAAGCACAACCAGAGCTGGCGGAAGCAGCCGCTAAAACCACCGAAAACCCGCTGCAGAAAATTGATGCCGCGCTGGCGCAGGTGGATGCGCTGCGCTCTGATCTGGGTGCGGTACAAAACCGTTTCAACTCTGCTATCACCAACCTGGGCAATACCGTAAACAATCTGTCTGAAGCGCGTAGCCGTATCGAAGATTCCGACTACGCGACCGAAGTTTCCAACATGTCTCGCGCGCAGATTCTGCAGCAGGCCGGTACTTCCGTTCTGGCGCAGGCTAACCAGGTCCCGCAGAACGTGCTGTCTCTGTTACGTAGCGGCAGTGGTAGCGGATCC STF2.HA1-1 PR8 SEQ ID NO: 53ATGGCACAAGTAATCAACACTAACAGTCTGTCGCTGCTGACCCAGAATAACCTGAACAAATCCCAGTCCGCACTGGGCACCGCTATCGAGCGTCTGTCTTCTGGTCTGCGTATCAACAGCGCGAAAGACGATGCGGCAGGTCAGGCGATTGCTAACCGTTTCACCGCGAACATCAAAGGTCTGACTCAGGCTTCCCGTAACGCTAACGACGGTATCTCCATTGCGCAGACCACTGAAGGCGCGCTGAACGAAATCAACAACAACCTGCAGCGTGTGCGTGAACTGGCGGTTCAGTCTGCTAACAGCACCAACTCCCAGTCTGACCTCGACTCCATCCAGGCTGAAATCACCCAGCGCCTGAACGAAATCGACCGTGTATCCGGCCAGACTCAGTTCAACGGCGTGAAAGTCCTGGCGCAGGACAACACCCTGACCATCCAGGTTGGCGCCAACGACGGTGAAACTATCGATATCGATCTGAAGCAGATCAACTCTCAGACCCTGGGTCTGGACTCACTGAACGTGCAGAAAGCGTATGATGTGAAAGATACAGCAGTAACAACGAAAGCTTATGCCAATAATGGTACTACACTGGATGTATCGGGTCTTGATGATGCAGCTATTAAAGCGGCTACGGGTGGTACGAATGGTACGGCTTCTGTAACCGGTGGTGCGGTTAAATTTGACGCAGATAATAACAAGTACTTTGTTACTATTGGTGGCTTTACTGGTGCTGATGCCGCCAAAAATGGCGATTATGAAGTTAACGTTGCTACTGACGGTACAGTAACCCTTGCGGCTGGCGCAACTAAAACCACAATGCCTGCTGGTGCGACAACTAAAACAGAAGTACAGGAGTTAAAAGATACACCGGCAGTTGTTTCAGCAGATGCTAAAAATGCCTTAATTGCTGGCGGCGTTGACGCTACCGATGCTAATGGCGCTGAGTTGGTCAAAATGTCTTATACCGATAAAAATGGTAAGACAATTGAAGGCGGTTATGCGCTTAAAGCTGGCGATAAGTATTACGCCGCAGATTACGATGAAGCGACAGGAGCAATTAAAGCTAAAACCACAAGTTATACTGCTGCTGACGGCACTACCAAAACAGCGGCTAACCAACTGGGTGGCGTAGACGGTAAAACCGAAGTCGTTACTATCGACGGTAAAACCTACAATGCCAGCAAAGCCGCTGGTCATGATTTCAAAGCACAACCAGAGCTGGCGGAAGCAGCCGCTAAAACCACCGAAAACCCGCTGCAGAAAATTGATGCCGCGCTGGCGCAGGTGGATGCGCTGCGCTCTGATCTGGGTGCGGTACAAAACCGTTTCAACTCTGCTATCACCAACCTGGGCAATACCGTAAACAATCTGTCTGAAGCGCGTAGCCGTATCGAAGATTCCGACTACGCGACCGAAGTTTCCAACATGTCTCGCGCGCAGATTTTGCAGCAGGCCGGTACTTCCGTTCTGGCGCAGGCTAACCAGGTCCCGCAGAACGTGCTGTCTCTGTTACGTAGCGGCAGTGGTAGCGGATCCTCTCATAACGGTAAACTGTGTCGTCTGAAAGGTATTGCACCACTGCAGCTGGGCAAATGCAACATCGCGGGTTGGCTGCTGGGTAACCCTGAATGTGACCCGCTGCTGCCGGTTCGTTCCTGGAGCTACATTGTTGAAACCCCGAACTCCGAAAACGGTATCTGCTACCCGGGCGACTTTATTGACTATGAAGAACTGCGTGAGCAGCTGTCTTCCGTGAGCAGCTTTGAACGCTTCGAAATCTTCCCGAAGGAAAGCTCCTGGCCGAACCACAACACTAACGGCGTGACGGCGGCTTGCTCCCACGAAGGCAAATCTTCCTTTTATCGTAACCTGCTGTGGCTGACTGAAAAGGAAGGTTCCTACCCAAAACTGAAAAACAGCTATGTTAACAAAAAGGGTAAAGAAGTCCTGGTGCTGTGGGGCATCCACCACCCGCCGAACTCCAAGGAACAGCAGAATCTGTATCAGAACGAAAACGCATACGTTTCTGTCGTTACTTCCAACTATAACCGTCGTTTCACTCCGGAAATCGCGGAACGTCCGAAAGTACGCGACCAGGCTGGCCGTATGAACTACTACTGGACCCTGCTGAAACCGGGTGACACTATTATCTTCGAAGCTAACGGTAACCTGATCGCACCAATGTACGCTTTCGCACTGTCTCGTGGTTTCGGTTCCGGCATTATCACCAGCAACGCGTCTATGCACGAATGCAACACGAAATGTCAGACGCCGCTGGGCGCAATTAATAGCAGCCTGCCGTACCAGAACATCCACCCGGTGACTATCGGCGAATGCCCGAAGTATGTTCGTTAATAG STF2.HA1-1.his PR8 SEQ ID NO: 54ATGGCACAAGTAATCAACACTAACAGTCTGTCGCTGCTGACCCAGAATAACCTGAACAAATCCCAGTCCGCACTGGGCACCGCTATCGAGCGTCTGTCTTCTGGTCTGCGTATCAACAGCGCGAAAGACGATGCGGCAGGTCAGGCGATTGCTAACCGTTTCACCGCGAACATCAAAGGTCTGACTCAGGCTTCCCGTAACGCTAACGACGGTATCTCCATTGCGCAGACCACTGAAGGCGCGCTGAACGAAATCAACAACAACCTGCAGCGTGTGCGTGAACTGGCGGTTCAGTCTGCTAACAGCACCAACTCCCAGTCTGACCTCGACTCCATCCAGGCTGAAATCACCCAGCGCCTGAACGAAATCGACCGTGTATCCGGCCAGACTCAGTTCAACGGCGTGAAAGTCCTGGCGCAGGACAACACCCTGACCATCCAGGTTGGCGCCAACGACGGTGAAACTATCGATATCGATCTGAAGCAGATCAACTCTCAGACCCTGGGTCTGGACTCACTGAACGTGCAGAAAGCGTATGATGTGAAAGATACAGCAGTAACAACGAAAGCTTATGCCAATAATGGTACTACACTGGATGTATCGGGTCTTGATGATGCAGCTATTAAAGCGGCTACGGGTGGTACGAATGGTACGGCTTCTGTAACCGGTGGTGCGGTTAAATTTGACGCAGATAATAACAAGTACTTTGTTACTATTGGTGGCTTTACTGGTGCTGATGCCGCCAAAAATGGCGATTATGAAGTTAACGTTGCTACTGACGGTACAGTAACCCTTGCGGCTGGCGCAACTAAAACCACAATGCCTGCTGGTGCGACAACTAAAACAGAAGTACAGGAGTTAAAAGATACACCGGCAGTTGTTTCAGCAGATGCTAAAAATGCCTTAATTGCTGGCGGCGTTGACGCTACCGATGCTAATGGCGCTGAGTTGGTCAAAATGTCTTATACCGATAAAAATGGTAAGACAATTGAAGGCGGTTATGCGCTTAAAGCTGGCGATAAGTATTACGCCGCAGATTACGATGAAGCGACAGGAGCAATTAAAGCTAAAACCACAAGTTATACTGCTGCTGACGGCACTACCAAAACAGCGGCTAACCAACTGGGTGGCGTAGACGGTAAAACCGAAGTCGTTACTATCGACGGTAAAACCTACAATGCCAGCAAAGCCGCTGGTCATGATTTCAAAGCACAACCAGAGCTGGCGGAAGCAGCCGCTAAAACCACCGAAAACCCGCTGCAAAAAATTGATGCCGCGCTGGCGCAGGTGGATGCGCTGCGCTCTGATCTGGGTGCGGTACAAAACCGTTTCAACTCTGCTATCACCAACCTGGGCAATACCGTAAACAATCTGTCTGAAGCGCGTAGCCGTATCGAAGATTCCGACTACGCGACCGAAGTTTCCAACATGTCTCGCGCGCAGATTTTGCAGCAGGCCGGTACTTCCGTTCTGGCGCAGGCTAACCAGGTCCCGCAGAACGTGCTGTCTCTGTTACGTAGCGGCAGTGGTAGCGGATCCTCTCATAACGGTAAACTGTGTCGTCTGAAAGGTATTGCACCACTGCAGCTGGGCAAATGCAACATCGCGGGTTGGCTGCTGGGTAACCCTGAATGTGACCCGCTGCTGCCGGTTCGTTCCTGGAGCTACATTGTTGAAACCCCGAACTCCGAAAACGGTATCTGCTACCCGGGCGACTTTATTGACTATGAAGAACTGCGTGAGCAGCTGTCTTCCGTGAGCAGCTTTGAACGCTTCGAAATCTTCCCGAAGGAAAGCTCCTGGCCGAACCACAACACTAACGGCGTGACGGCGGCTTGCTCCCACGAAGGCAAATCTTCCTTTTATCGTAACCTGCTGTGGCTGACTGAAAAGGAAGGTTCCTACCCAAAACTGAAAAACAGCTATGTTAACAAAAAGGGTAAAGAAGTCCTGGTGCTGTGGGGCATCCACCACCCGCCGAACTCCAAGGAACAGCAGAATCTGTATCAGAACGAAAACGCATACGTTTCTGTCGTTACTTCCAACTATAACCGTCGTTTCACTCCGGAAATCGCGGAACGTCCGAAAGTACGCGACCAGGCTGGCCGTATGAACTACTACTGGACCCTGCTGAAACCGGGTGACACTATTATCTTCGAAGCTAACGGTAACCTGATCGCACCAATGTACGCTTTCGCACTGTCTCGTGGTTTCGGTTCCGGCATTATCACCAGCAACGCGTCTATGCACGAATGCAACACGAAATGTCAGACGCCGCTGGGCGCAATTAATAGCAGCCTGCCGTACCAGAACATCCACCCGGTGACTATCGGCGAATGCCCGAAGTATGTTCGTCATCACCATCATCACCATTAATAG STF2.HA1-2 PR8 SEQ ID NO: 55ATGGCACAAGTAATCAACACTAACAGTCTGTCGCTGCTGACCCAGAATAACCTGAACAAATCCCAGTCCGCACTGGGCACCGCTATCGAGCGTCTGTCTTCTGGTCTGCGTATCAACAGCGCGAAAGACGATGCGGCAGGTCAGGCGATTGCTAACCGTTTCACCGCGAACATCAAAGGTCTGACTCAGGCTTCCCGTAACGCTAACGACGGTATCTCCATTGCGCAGACCACTGAAGGCGCGCTGAACGAAATCAACAACAACCTGCAGCGTGTGCGTGAACTGGCGGTTCAGTCTGCTAACAGCACCAACTCCCAGTCTGACCTCGACTCCATCCAGGCTGAAATCACCCAGCGCCTGAACGAAATCGACCGTGTATCCGGCCAGACTCAGTTCAACGGCGTGAAAGTCCTGGCGCAGGACAACACCCTGACCATCCAGGTTGGCGCCAACGACGGTGAAACTATCGATATCGATCTGAAGCAGATCAACTCTCAGACCCTGGGTCTGGACTCACTGAACGTGCAGAAAGCGTATGATGTGAAAGATACAGCAGTAACAACGAAAGCTTATGCCAATAATGGTACTACACTGGATGTATCGGGTCTTGATGATGCAGCTATTAAAGCGGCTACGGGTGGTACGAATGGTACGGCTTCTGTAACCGGTGGTGCGGTTAAATTTGACGCAGATAATAACAAGTACTTTGTTACTATTGGTGGCTTTACTGGTGCTGATGCCGCCAAAAATGGCGATTATGAAGTTAACGTTGCTACTGACGGTACAGTAACCCTTGCGGCTGGCGCAACTAAAACCACAATGCCTGCTGGTGCGACAACTAAAACAGAAGTACAGGAGTTAAAAGATACACCGGCAGTTGTTTCAGCAGATGCTAAAAATGCCTTAATTGCTGGCGGCGTTGACGCTACCGATGCTAATGGCGCTGAGTTGGTCAAAATGTCTTATACCGATAAAAATGGTAAGACAATTGAAGGCGGTTATGCGCTTAAAGCTGGCGATAAGTATTACGCCGCAGATTACGATGAAGCGACAGGAGCAATTAAAGCTAAAACTACAAGTTATACTGCTGCTGACGGCACTACCAAAACAGCGGCTAACCAACTGGGTGGCGTAGACGGTAAAACCGAAGTCGTTACTATCGACGGTAAAACCTACAATGCCAGCAAAGCCGCTGGTCATGATTTCAAAGCACAACCAGAGCTGGCGGAAGCAGCCGCTAAAACCACCGAAAACCCGCTGCAGAAAATTGATGCCGCGCTGGCGCAGGTGGATGCGCTGCGCTCTGATCTGGGTGCGGTACAAAACCGTTTCAACTCTGCTATCACCAACCTGGGCAATACCGTAAACAATCTGTCTGAAGCGCGTAGCCGTATCGAAGATTCCGACTACGCGACCGAAGTTTCCAACATGTCTCGCGCGCAGATTCTGCAGCAGGCCGGTACTTCCGTTCTGGCGCAGGCTAACCAGGTCCCGCAGAACGTGCTGTCTCTGTTACGTTCTGGTTCTGGTTCTGGTTCTAAAGGTATTGCTCCACTGCAACTGGGTAAATGCAATATTGCGGGTTGGCTGCTGGGCAACCCGGAATGCGATCCGCTGCTGCCGGTCCGTTCCTGGAGCTATATTGTTGAAACTCCGAACTCTGAGAACGGCATCTGCTATCCAGGTGATTTCATTGACTATGAGGAACTGCGTGAACAACTGTCTTCCGTGTCTTCCTTTGAACGTTTCGAGATTTTTCCTAAAGAATCTTCTTGGCCGAACCATAACACTAATGGTGTTACCGCTGCGTGCTCTCATGAAGGTAAATCTAGCTTTTACCGCAACCTGCTGTGGCTGACCGAGAAAGAAGGTTCTTACCCGAAACTGAAAAACAGCTACGTAAACAAAAAGGGCAAGGAAGTTCTGGTCCTGTGGGGTATCCACCATCCGCCGAACAGCAAGGAACAGCAGAATCTGTATCAGAACGAAAACGCATACGTATCTGTTGTTACTTCTAACTACAACCGCCGTTTCACCCCTGAAATCGCGGAACGTCCGAAAGTGCGTGACCAGGCAGGCCGCATGAACTATTACTGGACCCTGCTGAAGCCGGGTGATACTATCATCTTCGAAGCGAACGGTAACCTGATCGCCCCGATGTACGCGTTCGCTCTGAGCCGTGGCTTCGGCTCTGGTATCATTACGTCTTAATAA STF2.HA1-2mut PR8SEQ ID NO: 56ATGGCACAAGTAATCAACACTAACAGTCTGTCGCTGCTGACCCAGAATAACCTGAACAAATCCCAGTCCGCACTGGGCACCGCTATCGAGCGTCTGTCTTCTGGTCTGCGTATCAACAGCGCGAAAGACGATGCGGCAGGTCAGGCGATTGCTAACCGTTTCACCGCGAACATCAAAGGTCTGACTCAGGCTTCCCGTAACGCTAACGACGGTATCTCCATTGCGCAGACCACTGAAGGCGCGCTGAACGAAATCAACAACAACCTGCAGCGTGTGCGTGAACTGGCGGTTCAGTCTGCTAACAGCACCAACTCCCAGTCTGACCTCGACTCCATCCAGGCTGAAATCACCCAGCGCCTGAACGAAATCGACCGTGTATCCGGCCAGACTCAGTTCAACGGCGTGAAAGTCCTGGCGCAGGACAACACCCTGACCATCCAGGTTGGCGCCAACGACGGTGAAACTATCGATATCGATCTGAAGCAGATCAACTCTCAGACCCTGGGTCTGGACTCACTGAACGTGCAGAAAGCGTATGATGTGAAAGATACAGCAGTAACAACGAAAGCTTATGCCAATAATGGTACTACACTGGATGTATCGGGTCTTGATGATGCAGCTATTAAAGCGGCTACGGGTGGTACGAATGGTACGGCTTCTGTAACCGGTGGTGCGGTTAAATTTGACGCAGATAATAACAAGTACTTTGTTACTATTGGTGGCTTTACTGGTGCTGATGCCGCCAAAAATGGCGATTATGAAGTTAACGTTGCTACTGACGGTACAGTAACCCTTGCGGCTGGCGCAACTAAAACCACAATGCCTGCTGGTGCGACAACTAAAACAGAAGTACAGGAGTTAAAAGATACACCGGCAGTTGTTTCAGCAGATGCTAAAAATGCCTTAATTGCTGGCGGCGTTGACGCTACCGATGCTAATGGCGCTGAGTTGGTCAAAATGTCTTATACCGATAAAAATGGTAAGACAATTGAAGGCGGTTATGCGCTTAAAGCTGGCGATAAGTATTACGCCGCAGATTACGATGAAGCGACAGGAGCAATTAAAGCTAAAACCACAAGTTATACTGCTGCTGACGGCACTACCAAAACAGCGGCTAACCAACTGGGTGGCGTAGACGGTAAAACCGAAGTCGTTACTATCGACGGTAAAACCTACAATGCCAGCAAAGCCGCTGGTCATGATTTCAAAGCACAACCAGAGCTGGCGGAAGCAGCCGCTAAAACCACCGAAAACCCGCTGCAGAAAATTGATGCCGCGCTGGCGCAGGTGGATGCGCTGCGCTCTGATCTGGGTGCGGTACAAAACCGTTTCAACTCTGCTATCACCAACCTGGGCAATACCGTAAACAATCTGTCTGAAGCGCGTAGCCGTATCGAAGATTCCGACTACGCGACCGAAGTTTCCAACATGTCTCGCGCGCAGATTTTGCAGCAGGCCGGTACTTCCGTTCTGGCGCAGGCTAACCAGGTCCCGCAGAACGTGCTGTCTCTGTTAGCGAGCGGCAGTGGTAGCGGATCCAAAGGTGCGGCTCCACTGCAACTGGGTAAATGCAATATTGCGGGTTGGCTGCTGGGCAACCCGGAATGCGATCCGCTGCTGCCGGTCCGTTCCTGGAGCGATATTGCGGAAACTCCGAACTCTGAGAACGGCATCTGCTATCCAGGTGATTTCATTGACTATGAGGAACTGCGTGAACAACTGTCTTCCGTGTCTTCCTTTGAACGTTTCGAGATTTTTCCTAAAGAATCTTCTTGGCCGAACCATAACACTAATGGTGTTACCGCTGCGTGCTCTCATGAAGGTAAATCTAGCTTTTACCGCAACCTGCTGTGGCTGACCGAGAAAGAAGGTTCTTACCCGAAACTGAAAAACAGCTACGTAAACAAAAAGGGCAAGGAAGTTCTGGTCCTGTGGGGTATCCACCATCCGCCGAACAGCAAGGAACAGCAGAATCTGTATCAGAACGAAAACGCATACGTATCTGTTGTTACTTCTAACTACAACCGCCGTTTCACCCCTGAAATCGCGGAACGTCCGAAAGTGCGTGACCAGGCAGGCCGCATGAACTATTACTGGACCCTGCTGAAGCCGGGTGATACTATCATCTTCGAAGCGAACGGTAACCTGATCGCCCCGATGTACGCGTTCGCTCTGAGCCGTGGCTTCGGCTCTGGTATCATTACGTCTTAATAA STF2.HA1-3 PR8SEQ ID NO: 57ATGGCACAAGTAATCAACACTAACAGTCTGTCGCTGCTGACCCAGAATAACCTGAACAAATCCCAGTCCGCACTGGGCACCGCTATCGAGCGTCTGTCTTCTGGTCTGCGTATCAACAGCGCGAAAGACGATGCGGCAGGTCAGGCGATTGCTAACCGTTTCACCGCGAACATCAAAGGTCTGACTCAGGCTTCCCGTAACGCTAACGACGGTATCTCCATTGCGCAGACCACTGAAGGCGCGCTGAACGAAATCAACAACAACCTGCAGCGTGTGCGTGAACTGGCGGTTCAGTCTGCTAACAGCACCAACTCCCAGTCTGACCTCGACTCCATCCAGGCTGAAATCACCCAGCGCCTGAACGAAATCGACCGTGTATCCGGCCAGACTCAGTTCAACGGCGTGAAAGTCCTGGCGCAGGACAACACCCTGACCATCCAGGTTGGCGCCAACGACGGTGAAACTATCGATATCGATCTGAAGCAGATCAACTCTCAGACCCTGGGTCTGGACTCACTGAACGTGCAGAAAGCGTATGATGTGAAAGATACAGCAGTAACAACGAAAGCTTATGCCAATAATGGTACTACACTGGATGTATCGGGTCTTGATGATGCAGCTATTAAAGCGGCTACGGGTGGTACGAATGGTACGGCTTCTGTAACCGGTGGTGCGGTTAAATTTGACGCAGATAATAACAAGTACTTTGTTACTATTGGTGGCTTTACTGGTGCTGATGCCGCCAAAAATGGCGATTATGAAGTTAACGTTGCTACTGACGGTACAGTAACCCTTGCGGCTGGCGCAACTAAAACCACAATGCCTGCTGGTGCGACAACTAAAACAGAAGTACAGGAGTTAAAAGATACACCGGCAGTTGTTTCAGCAGATGCTAAAAATGCCTTAATTGCTGGCGGCGTTGACGCTACCGATGCTAATGGCGCTGAGTTGGTCAAAATGTCTTATACCGATAAAAATGGTAAGACAATTGAAGGCGGTTATGCGCTTAAAGCTGGCGATAAGTATTACGCCGCAGATTACGATGAAGCGACAGGAGCAATTAAAGCTAAAACTACAAGTTATACTGCTGCTGACGGCACTACCAAAACAGCGGCTAACCAACTGGGTGGCGTAGACGGTAAAACCGAAGTCGTTACTATCGACGGTAAAACCTACAATGCCAGCAAAGCCGCTGGTCATGATTTCAAAGCACAACCAGAGCTGGCGGAAGCAGCCGCTAAAACCACCGAAAACCCGCTGCAGAAAATTGATGCCGCGCTGGCGCAGGTGGATGCGCTGCGCTCTGATCTGGGTGCGGTACAAAACCGTTTCAACTCTGCTATCACCAACCTGGGCAATACCGTAAACAATCTGTCTGAAGCGCGTAGCCGTATCGAAGATTCCGACTACGCGACCGAAGTTTCCAACATGTCTCGCGCGCAGATTCTGCAGCAGGCCGGTACTTCCGTTCTGGCGCAGGCTAACCAGGTCCCGCAGAACGTGCTGTCTCTGTTACGTTCTGGTAGCGGTTCTGGCTCTAACTCTGAAAATGGTATCTGTTACCCGGGTGATTTCATCGATTATGAGGAACTGCGTGAACAGCTGTCTAGCGTGAGCTCCTTTGAACGCTTCGAAATCTTCCCGAAGGAATCTAGCTGGCCGAACCATAACACGAACGGTGTGACCGCTGCTTGCTCCCACGAAGGTAAGAGCTCCTTCTACCGCAATCTGCTGTGGCTGACTGAAAAAGAAGGTAGCTACCCGAAACTGAAAAATTCTTACGTCAACAAGAAAGGCAAGGAAGTGCTGGTTCTGTGGGGCATTCACCACCCACCGAACAGCAAAGAGCAACAGAACCTGTACCAAAATGAGAACGCTTACGTTTCTGTTGTGACTTCTAACTACAATCGTCGCTTTACCCCTGAAATCGCGGAGCGTCCAAAAGTGCGTGACCAGGCTGGTCGTATGAACTACTATTGGACCCTGCTGAAACCGGGCGACACCATTATCTTTGAAGCGAACGGTAACCTGATCGCGCCTATGTACGCGTTCGCTCTGTCTCGTGGCTAATAA STF2.HA1-3mut PR8 SEQ ID NO: 58ATGGCACAAGTAATCAACACTAACAGTCTGTCGCTGCTGACCCAGAATAACCTGAACAAATCCCAGTCCGCACTGGGCACCGCTATCGAGCGTCTGTCTTCTGGTCTGCGTATCAACAGCGCGAAAGACGATGCGGCAGGTCAGGCGATTGCTAACCGTTTCACCGCGAACATCAAAGGTCTGACTCAGGCTTCCCGTAACGCTAACGACGGTATCTCCATTGCGCAGACCACTGAAGGCGCGCTGAACGAAATCAACAACAACCTGCAGCGTGTGCGTGAACTGGCGGTTCAGTCTGCTAACAGCACCAACTCCCAGTCTGACCTCGACTCCATCCAGGCTGAAATCACCCAGCGCCTGAACGAAATCGACCGTGTATCCGGCCAGACTCAGTTCAACGGCGTGAAAGTCCTGGCGCAGGACAACACCCTGACCATCCAGGTTGGCGCCAACGACGGTGAAACTATCGATATCGATCTGAAGCAGATCAACTCTCAGACCCTGGGTCTGGACTCACTGAACGTGCAGAAAGCGTATGATGTGAAAGATACAGCAGTAACAACGAAAGCTTATGCCAATAATGGTACTACACTGGATGTATCGGGTCTTGATGATGCAGCTATTAAAGCGGCTACGGGTGGTACGAATGGTACGGCTTCTGTAACCGGTGGTGCGGTTAAATTTGACGCAGATAATAACAAGTACTTTGTTACTATTGGTGGCTTTACTGGTGCTGATGCCGCCAAAAATGGCGATTATGAAGTTAACGTTGCTACTGACGGTACAGTAACCCTTGCGGCTGGCGCAACTAAAACCACAATGCCTGCTGGTGCGACAACTAAAACAGAAGTACAGGAGTTAAAAGATACACCGGCAGTTGTTTCAGCAGATGCTAAAAATGCCTTAATTGCTGGCGGCGTTGACGCTACCGATGCTAATGGCGCTGAGTTGGTCAAAATGTCTTATACCGATAAAAATGGTAAGACAATTGAAGGCGGTTATGCGCTTAAAGCTGGCGATAAGTATTACGCCGCAGATTACGATGAAGCGACAGGAGCAATTAAAGCTAAAACTACAAGTTATACTGCTGCTGACGGCACTACCAAAACAGCGGCTAACCAACTGGGTGGCGTAGACGGTAAAACCGAAGTCGTTACTATCGACGGTAAAACCTACAATGCCAGCAAAGCCGCTGGTCATGATTTCAAAGCACAACCAGAGCTGGCGGAAGCAGCCGCTAAAACCACCGAAAACCCGCTGCAGAAAATTGATGCCGCGCTGGCGCAGGTGGATGCGCTGCGCTCTGATCTGGGTGCGGTACAAAACCGTTTCAACTCTGCTATCACCAACCTGGGCAATACCGTAAACAATCTGTCTGAAGCGCGTAGCCGTATCGAAGATTCCGACTACGCGACCGAAGTTTCCAACATGTCTCGCGCGCAGATTCTGCAGCAGGCCGGTACTTCCGTTCTGGCGCAGGCTAACCAGGTCCCGCAGAACGTGCTGTCTCTGTTACGTAGCGGCAGTGGTAGCGGATCCAACTCTGAAAATGAAATCTGTTACCCGGGTGATTTCATCGATAAAGAGGAACTGCGTGAACAGCTGTCTAGCGTGAGCTCCTTTGAACGCTTCGAAATCTTCCCGAAGGAATCTAGCTGGCCGAACCATAACACGAACGGTGTGACCGCTGCTTGCTCCCACGAAGGTAAGAGCTCCTTCTACCGCAATCTGCTGTGGCTGACTGAAAAAGAAGGTAGCTACCCGAAACTGAAAAATTCTTACGTCAACAAGAAAGGCAAGGAAGTGCTGGTTCTGTGGGGCATTCACCACCCACCGAACAGCAAAGAGCAACAGAACCTGTACCAAAATGAGAACGCTTACGTTTCTGTTGTGACTTCTAACTACAATCGTCGCTTTACCCCTGAAATCGCGGAGCGTCCAAAAGTGCGTGACCAGGCTGGTCGTATGAACTACTATTGGACCCTGCTGAAACCGGGCGACACCATTATCTTTGAAGCGAACGGTAACCTGATCGCGCCTATGTACGCGGCGGCTCTGTCTCGTGGCTAATAA HA1-1 PR8 SEQ ID NO: 59ATGTCTCATAACGGTAAACTGTGTCGTCTGAAAGGTATTGCACCACTGCAGCTGGGCAAATGCAACATCGCGGGTTGGCTGCTGGGTAACCCTGAATGTGACCCGCTGCTGCCGGTTCGTTCCTGGAGCTACATTGTTGAAACCCCGAACTCCGAAAACGGTATCTGCTACCCGGGCGACTTTATTGACTATGAAGAACTGCGTGAGCAGCTGTCTTCCGTGAGCAGCTTTGAACGCTTCGAAATCTTCCCGAAGGAAAGCTCCTGGCCGAACCACAACACTAACGGCGTGACGGCGGCTTGCTCCCACGAAGGCAAATCTTCCTTTTATCGTAACCTGCTGTGGCTGACTGAAAAGGAAGGTTCCTACCCAAAACTGAAAAACAGCTATGTTAACAAAAAGGGTAAAGAAGTCCTGGTGCTGTGGGGCATCCACCACCCGCCGAACTCCAAGGAACAGCAGAATCTGTATCAGAACGAAAACGCATACGTTTCTGTCGTTACTTCCAACTATAACCGTCGTTTCACTCCGGAAATCGCGGAACGTCCGAAAGTACGCGACCAGGCTGGCCGTATGAACTACTACTGGACCCTGCTGAAACCGGGTGACACTATTATCTTCGAAGCTAACGGTAACCTGATCGCACCAATGTACGCTTTCGCACTGTCTCGTGGTTTCGGTTCCGGCATTATCACCAGCAACGCGTCTATGCACGAATGCAACACGAAATGTCAGACGCCGCTGGGCGCAATTAATAGCAGCCTGCCGTACCAGAACATCCACCCGGTGACTATCGGCGAATGCCCGAAGTATGTTCGT HA1-1.his PR8 SEQ ID NO: 60ATGTCTCATAACGGTAAACTGTGTCGTCTGAAAGGTATTGCACCACTGCAGCTGGGCAAATGCAACATCGCGGGTTGGCTGCTGGGTAACCCTGAATGTGACCCGCTGCTGCCGGTTCGTTCCTGGAGCTACATTGTTGAAACCCCGAACTCCGAAAACGGTATCTGCTACCCGGGCGACTTTATTGACTATGAAGAACTGCGTGAGCAGCTGTCTTCCGTGAGCAGCTTTGAACGCTTCGAAATCTTCCCGAAGGAAAGCTCCTGGCCGAACCACAACACTAACGGCGTGACGGCGGCTTGCTCCCACGAAGGCAAATCTTCCTTTTATCGTAACCTGCTGTGGCTGACTGAAAAGGAAGGTTCCTACCCAAAACTGAAAAACAGCTATGTTAACAAAAAGGGTAAAGAAGTCCTGGTGCTGTGGGGCATCCACCACCCGCCGAACTCCAAGGAACAGCAGAATCTGTATCAGAACGAAAACGCATACGTTTCTGTCGTTACTTCCAACTATAACCGTCGTTTCACTCCGGAAATCGCGGAACGTCCGAAAGTACGCGACCAGGCTGGCCGTATGAACTACTACTGGACCCTGCTGAAACCGGGTGACACTATTATCTTCGAAGCTAACGGTAACCTGATCGCACCAATGTACGCTTTCGCACTGTCTCGTGGTTTCGGTTCCGGCATTATCACCAGCAACGCGTCTATGCACGAATGCAACACGAAATGTCAGACGCCGCTGGGCGCAATTAATAGCAGCCTGCCGTACCAGAACATCCACCCGGTGACTATCGGCGAATGCCCGAAGTATGTTCGTCATCACCATCATCACCAT HA1-2.his PR8SEQ ID NO: 61ATGAAAGGTATTGCTCCACTGCAACTGGGTAAATGCAATATTGCGGGTTGGCTGCTGGGCAACCCGGAATGCGATCCGCTGCTGCCGGTCCGTTCCTGGAGCTATATTGTTGAAACTCCGAACTCTGAGAACGGCATCTGCTATCCAGGTGATTTCATTGACTATGAGGAACTGCGTGAACAACTGTCTTCCGTGTCTTCCTTTGAACGTTTCGAGATTTTTCCTAAAGAATCTTCTTGGCCGAACCATAACACTAATGGTGTTACCGCTGCGTGCTCTCATGAAGGTAAATCTAGCTTTTACCGCAACCTGCTGTGGCTGACCGAGAAAGAAGGTTCTTACCCGAAACTGAAAAACAGCTACGTAAACAAAAAGGGCAAGGAAGTTCTGGTCCTGTGGGGTATCCACCATCCGCCGAACAGCAAGGAACAGCAGAATCTGTATCAGAACGAAAACGCATACGTATCTGTTGTTACTTCTAACTACAACCGCCGTTTCACCCCTGAAATCGCGGAACGTCCGAAAGTGCGTGACCAGGCAGGCCGCATGAACTATTACTGGACCCTGCTGAAGCCGGGTGATACTATCATCTTCGAAGCGAACGGTAACCTGATCGCCCCGATGTACGCGTTCGCTCTGAGCCGTGGCTTCGGCTCTGGTATCATTACGTCTCATCACCATCATCACCAT CRM.HA1-2 SEQ ID NO: 62ATGGGTGCTGATGATGTCGTTGATTCCTCCAAAAGCTTCGTTATGGAAAATTTCTCTTCTTATCACGGCACCAAACCGGGTTATGTGGATTCTATCCAAAAAGGCATCCAGAAACCGAAGTCCGGTACGCAGGGTAACTATGATGACGATTGGAAAGGTTTTTACTCCACCGATAATAAATATGACGCGGCTGGCTACTCTGTTGACAACGAAAATCCACTGTCTGGTAAGGCTGGCGGTGTCGTAAAGGTAACGTATCCTGGCCTGACCAAGGTGCTGGCACTGAAGGTGGATAACGCTGAAACCATCAAAAAGGAGCTGGGCCTGAGCCTGACTGAACCGCTGATGGAGCAGGTCGGTACCGAGGAATTCATCAAACGCTTCGGTGATGGCGCCTCCCGTGTTGTGCTGTCCCTGCCGTTCGCAGAAGGCTCTTCCTCTGTCGAATATATCAACAACTGGGAACAGGCTAAAGCTCTGAGCGTCGAACTGGAAATTAACTTTGAGACCCGTGGCAAGCGTGGTCAGGACGCGATGTACGAATATATGGCTCAGGCTTGTGCCGGTAACCGTGTTCGCCGTTCCGTCGGTTCCTCTCTGTCTTGCATCAACCTGGATTGGGACGTTATCCGTGATAAGACCAAAACCAAAATTGAAAGCCTGAAGGAACACGGTCCGATCAAAAACAAAATGTCTGAATCTCCGAACAAAACCGTGTCCGAGGAAAAAGCGAAACAGTATCTGGAAGAATTCCACCAGACTGCCCTGGAACATCCTGAACTGTCCGAACTGAAGACTGTAACCGGCACTAACCCGGTGTTCGCAGGCGCAAACTACGCCGCGTGGGCGGTAAACGTTGCGCAGGTTATTGATAGCGAAACCGCAGATAACCTGGAAAAAACGACCGCAGCTCTGTCTATCCTGCCGGGTATCGGTTCCGTTATGGGTATTGCGGACGGCGCTGTGCACCACAACACGGAAGAAATCGTCGCACAGTCTATCGCGCTGTCTTCTCTGATGGTTGCTCAGGCAATTCCACTGGTAGGTGAACTGGTGGACATTGGCTTTGCGGCGTACAACTTCGTCGAAAGCATTATCAACCTGTTCCAGGTTGTACACAACTCTTACAACCGTCCGGCCTACAGCCCTGGCCACAAAACCCAACCGTTTCTGCACGACGGTTATGCGGTGTCCTGGAACACGGTTGAAGACTCTATCATTCGTACCGGCTTTCAGGGCGAGTCCGGCCACGATATCAAAATTACTGCAGAAAACACTCCGCTGCCGATCGCTGGCGTTCTGCTGCCGACCATCCCGGGTAAGCTGGATGTGAACAAAAGCAAAACCCACATCTCTGTTAACGGTCGTAAAATTCGCATGCGCTGTCGTGCTATCGACGGTGATGTTACCTTCTGCCGTCCGAAATCTCCAGTCTACGTGGGCAACGGTGTTCATGCCAACCTGCACGTGGCGTTCCATCGTAGCTCTAGCGAAAAAATCCACTCCAACGAAATCTCTAGCGATTCTATCGGTGTTCTGGGTTATCAGAAAACGGTGGATCATACGAAAGTCAATTCTAAACTGAGCCTGTTCTTCGAAATCAAATCTAGCGGCTCTGGATCCGGTTCCAAAGGCATCGCGCCGCTGCAGCTGGGTAAATGTAACATTGCGGGCTGGCTGCTGGGTAATCCGGAATGCGATCCGCTGCTGCCGGTCCGTAGCTGGTCTTACATTGTTGAAACTCCGAACTCTGAGAATGGCATCTGCTACCCGGGCGATTTTATCGACTATGAAGAACTGCGTGAACAGCTGTCTTCCGTTTCTTCCTTTGAACGTTTCGAAATCTTCCCGAAAGAAAGCAGCTGGCCGAATCACAATACGAACGGTGTTACTGCTGCGTGTTCTCATGAAGGTAAATCCAGCTTCTACCGTAACCTGCTGTGGCTGACCGAAAAAGAGGGTTCTTATCCTAAACTGAAAAACAGCTACGTTAACAAAAAGGGCAAAGAAGTGCTGGTGCTGTGGGGTATCCATCACCCTCCGAACTCTAAAGAACAACAGAATCTGTATCAGAACGAAAACGCTTACGTTTCCGTGGTGACCTCTAACTATAACCGTCGTTTTACCCCGGAGATTGCTGAACGTCCGAAAGTGCGCGATCAGGCTGGCCGTATGAACTACTATTGGACCCTGCTGAAACCGGGCGATACCATCATTTTCGAAGCTAACGGCAACCTGATTGCTCCGATGTATGCGTTTGCTCTGTCTCGTGGCTTCGGCTCTGGTATTATTACGTCTTAATAA LTB.HA1-2 SEQ ID NO: 63ATGAATAAGGTTAAGTTCTACGTACTGTTTACCGCGCTGCTGTCTTCTCTGTGCGCGCATGGTGCTCCGCAGTCTATTACTGAACTGTGCTCTGAATACCACAACACCCAGATCTATACTATCAACGATAAAATCCTGAGCTATACCGAATCTATGGCAGGCAAACGCGAAATGGTTATCATTACCTTTAAAAGCGGCGCCACCTTTCAAGTGGAAGTTCCGGGCTCTCAGCATATTGACTCTCAAAAAAAAGCGATCGAACGTATGAAAGATACTCTGCGCATTACCTACCTGACCGAAACCAAAATCGATAAACTGTGCGTATGGAACAATAAGACTCCTAACTCTATCGCAGCTATTTCTATGGAAAACTCTGGTAGCGGATCCGGTTCTAAAGGCATCGCGCCGCTGCAGCTGGGTAAATGTAACATTGCGGGCTGGCTGCTGGGTAATCCGGAATGCGATCCGCTGCTGCCGGTCCGTAGCTGGTCTTACATTGTTGAAACTCCGAACTCTGAGAATGGCATCTGCTACCCGGGCGATTTTATCGACTATGAAGAACTGCGTGAACAGCTGTCTTCCGTTTCTTCCTTTGAACGTTTCGAAATCTTCCCGAAAGAAAGCAGCTGGCCGAATCACAATACGAACGGTGTTACTGCTGCGTGTTCTCATGAAGGTAAATCCAGCTTCTACCGTAACCTGCTGTGGCTGACCGAAAAAGAGGGTTCTTATCCTAAACTGAAAAACAGCTACGTTAACAAAAAGGGCAAAGAAGTGCTGGTGCTGTGGGGTATCCATCACCCTCCGAACTCTAAAGAACAACAGAATCTGTATCAGAACGAAAACGCTTACGTTTCCGTGGTGACCTCTAACTATAACCGTCGTTTTACCCCGGAGATTGCTGAACGTCCGAAAGTGCGCGATCAGGCTGGCCGTATGAACTACTATTGGACCCTGCTGAAACCGGGCGATACCATCATTTTCGAAGCTAACGGCAACCTGATTGCTCCGATGTATGCGTTTGCTCTGTCTCGTGGCTTCGGCTCTGGTATTATTACGTCTTAATAA STF2.HA1-1 VNSEQ ID NO: 64ATGGCACAAGTAATCAACACTAACAGTCTGTCGCTGCTGACCCAGAATAACCTGAACAAATCCCAGTCCGCACTGGGCACCGCTATCGAGCGTCTGTCTTCTGGTCTGCGTATCAACAGCGCGAAAGACGATGCGGCAGGTCAGGCGATTGCTAACCGTTTCACCGCGAACATCAAAGGTCTGACTCAGGCTTCCCGTAACGCTAACGACGGTATCTCCATTGCGCAGACCACTGAAGGCGCGCTGAACGAAATCAACAACAACCTGCAGCGTGTGCGTGAACTGGCGGTTCAGTCTGCTAACAGCACCAACTCCCAGTCTGACCTCGACTCCATCCAGGCTGAAATCACCCAGCGCCTGAACGAAATCGACCGTGTATCCGGCCAGACTCAGTTCAACGGCGTGAAAGTCCTGGCGCAGGACAACACCCTGACCATCCAGGTTGGCGCCAACGACGGTGAAACTATCGATATCGATCTGAAGCAGATCAACTCTCAGACCCTGGGTCTGGACTCACTGAACGTGCAGAAAGCGTATGATGTGAAAGATACAGCAGTAACAACGAAAGCTTATGCCAATAATGGTACTACACTGGATGTATCGGGTCTTGATGATGCAGCTATTAAAGCGGCTACGGGTGGTACGAATGGTACGGCTTCTGTAACCGGTGGTGCGGTTAAATTTGACGCAGATAATAACAAGTACTTTGTTACTATTGGTGGCTTTACTGGTGCTGATGCCGCCAAAAATGGCGATTATGAAGTTAACGTTGCTACTGACGGTACAGTAACCCTTGCGGCTGGCGCAACTAAAACCACAATGCCTGCTGGTGCGACAACTAAAACAGAAGTACAGGAGTTAAAAGATACACCGGCAGTTGTTTCAGCAGATGCTAAAAATGCCTTAATTGCTGGCGGCGTTGACGCTACCGATGCTAATGGCGCTGAGTTGGTCAAAATGTCTTATACCGATAAAAATGGTAAGACAATTGAAGGCGGTTATGCGCTTAAAGCTGGCGATAAGTATTACGCCGCAGATTACGATGAAGCGACAGGAGCAATTAAAGCTAAAACCACAAGTTATACTGCTGCTGACGGCACTACCAAAACAGCGGCTAACCAACTGGGTGGCGTAGACGGTAAAACCGAAGTCGTTACTATCGACGGTAAAACCTACAATGCCAGCAAAGCCGCTGGTCATGATTTCAAAGCACAACCAGAGCTGGCGGAAGCAGCCGCTAAAACCACCGAAAACCCGCTGCAGAAAATTGATGCCGCGCTGGCGCAGGTGGATGCGCTGCGCTCTGATCTGGGTGCGGTACAAAACCGTTTCAACTCTGCTATCACCAACCTGGGCAATACCGTAAACAATCTGTCTGAAGCGCGTAGCCGTATCGAAGATTCCGACTACGCGACCGAAGTTTCCAACATGTCTCGCGCGCAGATTTTGCAGCAGGCCGGTACTTCCGTTCTGGCGCAGGCTAACCAGGTCCCGCAGAACGTGCTGAGCCTGTTAGCGGAGAAGAAACACAATGGCAAACTGTGTGATCTGGATGGTGTGAAACCGCTGATTCTGCGCGATTGCTCTGTGGCAGGCTGGCTGCTGGGCAACCCTATGTGTGACGAATTCATTAACGTTCCGGAATGGTCTTACATTGTTGAAAAAGCTAACCCTGTCAACGATCTGTGTTACCCTGGTGACTTTAACGATTACGAAGAACTGAAGCACCTGCTGTCTCGTATCAATCACTTCGAGAAAATCCAGATCATCCCGAAATCCTCCTGGAGCTCCCACGAAGCTTCTCTGGGCGTATCCTCCGCGTGCCCGTACCAGGGCAAATCCTCTTTCTTTCGTAACGTTGTTTGGCTGATCAAGAAAAACTCCACCTACCCGACGATCAAGCGTAGCTATAATAACACCAACCAGGAAGACCTGCTGGTTCTGTGGGGCATCCACCATCCAAACGATGCTGCGGAACAGACCAAGCTGTACCAGAACCCGACCACCTACATCAGCGTGGGCACCTCTACGCTGAACCAGCGTCTGGTACCGCGTATCGCAACCCGCAGCAAGGTAAACGGTCAAAGCGGCCGCATGGAATTTTTCTGGACCATCCTGAAACCGAACGACGCAATCAACTTCGAATCTAACGGCAATTTCATCGCTCCGGAGTATGCGTACAAAATCGTAAAGAAAGGTGATAGCACTATCATGAAATCCGAGCTGGAATATGGCAACTGTAACACCAAATGCCAGACCCCGATGGGTGCAATCAACTCCTCCATGCCGTTTCACAACATTCACCCGCTGACTATCGGCGAATGTCCGAAATACGTTAAA TAGTAAFOR PRIMER SEQ ID NO: 65 AGTGGCTGAGCCTGTTAGCGGAGAAGAAACACAATGGCAAACTGTGREV PRIMER SEQ ID NO: 66AGTCGCTCAGCTTACTATTTAACGTATTTCGGACATTCGCCGATAGTCAG HA0s VN SEQ ID NO: 67GTGCTGAGCCTGTTACGTCAGATTTGTATCGGCTACCACGCAAACAACTCTACCGAGCAAGTTGATACCATCATGGAGAAAAACGTGACCGTTACTCACGCGCAGGACATCCTGGAGAAGAAACACAATGGCAAACTGTGTGATCTGGATGGTGTGAAACCGCTGATTCTGCGCGATTGCTCTGTGGCAGGCTGGCTGCTGGGCAACCCTATGTGTGACGAATTCATTAACGTTCCGGAATGGTCTTACATTGTTGAAAAAGCTAACCCTGTCAACGATCTGTGTTACCCTGGTGACTTTAACGATTACGAAGAACTGAAGCACCTGCTGTCTCGTATCAATCACTTCGAGAAAATCCAGATCATCCCGAAATCCTCCTGGAGCTCCCACGAAGCTTCTCTGGGCGTATCCTCCGCGTGCCCGTACCAGGGCAAATCCTCTTTCTTTCGTAACGTTGTTTGGCTGATCAAGAAAAACTCCACCTACCCGACGATCAAGCGTAGCTATAATAACACCAACCAGGAAGACCTGCTGGTTCTGTGGGGCATCCACCATCCAAACGATGCTGCGGAACAGACCAAGCTGTACCAGAACCCGACCACCTACATCAGCGTGGGCACCTCTACGCTGAACCAGCGTCTGGTACCGCGTATCGCAACCCGCAGCAAGGTAAACGGTCAAAGCGGCCGCATGGAATTTTTCTGGACCATCCTGAAACCGAACGACGCAATCAACTTCGAATCTAACGGCAATTTCATCGCTCCGGAGTATGCGTACAAAATCGTAAAGAAAGGTGATAGCACTATCATGAAATCCGAGCTGGAATATGGCAACTGTAACACCAAATGCCAGACCCCGATGGGTGCAATCAACTCCTCCATGCCGTTTCACAACATTCACCCGCTGACTATCGGCGAATGTCCGAAATACGTTAAATCCAATCGTCTGGTTCTGGCTACCGGTCTGCGTAACTCCCCACAGCGTGAACGTCGTCGTAAGAAACGTGGTCTGTTTGGCGCGATCGCTGGTTTCATCGAGGGCGGCTGGCAGGGTATGGTTGATGGCTGGTACGGTTATCATCATTCCAATGAACAGGGTTCCGGCTACGCCGCAGATAAAGAAAGCACTCAGAAAGCAATTGATGGCGTAACTAACAAAGTAAATTCTATCATTGATAAAATGAACACCCAGTTCGAGGCGGTTGGTCGTGAGTTCAACAACCTGGAACGCCGTATCGAAAACCTGAACAAGAAAATGGAAGACGGTTTTCTGGATGTGTGGACTTACAATGCTGAACTGCTGGTGCTGATGGAAAACGAGCGTACCCTGGACTTCCACGACAGCAACGTCAAAAATCTGTATGACAAAGTCCGTCTGCAGCTGCGTGATAACGCTAAAGAGCTGGGTAATGGCTGCTTCGAGTTCTATCACAAATGCGACAACGAATGCATGGAATCTGTCCGCAACGGCACTTACGATTATCCGCAGTACTCCGAAGAAGCGCGCCTGAAACGCGAAGAGATCTCCGGTGTGAAGCTGGAGTCTATTGGCATCTACCAGATCCTGTCCATCTACAGCACCTAGTAAGCTGAGCGCCTACGCAGC STF2.HA1-2 VNSEQ ID NO: 68ATGGCACAAGTAATCAACACTAACAGTCTGTCGCTGCTGACCCAGAATAACCTGAACAAATCCCAGTCCGCACTGGGCACCGCTATCGAGCGTCTGTCTTCTGGTCTGCGTATCAACAGCGCGAAAGACGATGCGGCAGGTCAGGCGATTGCTAACCGTTTCACCGCGAACATCAAAGGTCTGACTCAGGCTTCCCGTAACGCTAACGACGGTATCTCCATTGCGCAGACCACTGAAGGCGCGCTGAACGAAATCAACAACAACCTGCAGCGTGTGCGTGAACTGGCGGTTCAGTCTGCTAACAGCACCAACTCCCAGTCTGACCTCGACTCCATCCAGGCTGAAATCACCCAGCGCCTGAACGAAATCGACCGTGTATCCGGCCAGACTCAGTTCAACGGCGTGAAAGTCCTGGCGCAGGACAACACCCTGACCATCCAGGTTGGCGCCAACGACGGTGAAACTATCGATATCGATCTGAAGCAGATCAACTCTCAGACCCTGGGTCTGGACTCACTGAACGTGCAGAAAGCGTATGATGTGAAAGATACAGCAGTAACAACGAAAGCTTATGCCAATAATGGTACTACACTGGATGTATCGGGTCTTGATGATGCAGCTATTAAAGCGGCTACGGGTGGTACGAATGGTACGGCTTCTGTAACCGGTGGTGCGGTTAAATTTGACGCAGATAATAACAAGTACTTTGTTACTATTGGTGGCTTTACTGGTGCTGATGCCGCCAAAAATGGCGATTATGAAGTTAACGTTGCTACTGACGGTACAGTAACCCTTGCGGCTGGCGCAACTAAAACCACAATGCCTGCTGGTGCGACAACTAAAACAGAAGTACAGGAGTTAAAAGATACACCGGCAGTTGTTTCAGCAGATGCTAAAAATGCCTTAATTGCTGGCGGCGTTGACGCTACCGATGCTAATGGCGCTGAGTTGGTCAAAATGTCTTATACCGATAAAAATGGTAAGACAATTGAAGGCGGTTATGCGCTTAAAGCTGGCGATAAGTATTACGCCGCAGATTACGATGAAGCGACAGGAGCAATTAAAGCTAAAACCACAAGTTATACTGCTGCTGACGGCACTACCAAAACAGCGGCTAACCAACTGGGTGGCGTAGACGGTAAAACCGAAGTCGTTACTATCGACGGTAAAACCTACAATGCCAGCAAAGCCGCTGGTCATGATTTCAAAGCACAACCAGAGCTGGCGGAAGCAGCCGCTAAAACCACCGAAAACCCGCTGCAGAAAATTGATGCCGCGCTGGCGCAGGTGGATGCGCTGCGCTCTGATCTGGGTGCGGTACAAAACCGTTTCAACTCTGCTATCACCAACCTGGGCAATACCGTAAACAATCTGTCTGAAGCGCGTAGCCGTATCGAAGATTCCGACTACGCGACCGAAGTTTCCAACATGTCTCGCGCGCAGATTTTGCAGCAGGCCGGTACTTCCGTTCTGGCGCAGGCTAACCAGGTCCCGCAGAACGTGCTGAGCCTGTTAGCAGGTGTGAAACCGCTGATTCTGCGCGATTGCTCTGTGGCAGGCTGGCTGCTGGGCAACCCTATGTGTGACGAATTCATTAACGTTCCGGAATGGTCTTACATTGTTGAAAAAGCTAACCCTGTCAACGATCTGTGTTACCCTGGTGACTTTAACGATTACGAAGAACTGAAGCACCTGCTGTCTCGTATCAATCACTTCGAGAAAATCCAGATCATCCCGAAATCCTCCTGGAGCTCCCACGAAGCTTCTCTGGGCGTATCCTCCGCGTGCCCGTACCAGGGCAAATCCTCTTTCTTTCGTAACGTTGTTTGGCTGATCAAGAAAAACTCCACCTACCCGACGATCAAGCGTAGCTATAATAACACCAACCAGGAAGACCTGCTGGTTCTGTGGGGCATCCACCATCCAAACGATGCTGCGGAACAGACCAAGCTGTACCAGAACCCGACCACCTACATCAGCGTGGGCACCTCTACGCTGAACCAGCGTCTGGTACCGCGTATCGCAACCCGCAGCAAGGTAAACGGTCAAAGCGGCCGCATGGAATTTTTCTGGACCATCCTGAAACCGAACGACGCAATCAACTTCGAATCTAACGGCAATTTCATCGCTCCGGAGTATGCGTACAAAATCGTAAAGAAAGGTGATAGCACTATCATGAAATCCGAGTAGTAA FOR PRIMER SEQ ID NO: 69AGTCGCTGAGCCTGTTAGCAGGTGTGAAACCGCTGATTCTGCGCGATTG REV PRIMERSEQ ID NO: 70 TGACGCTCAGCTTACTACTCGGATTTCATGATAGTGCTATCACCTTTCSTF2.HA1-2mut VN SEQ ID NO: 71ATGGCACAAGTAATCAACACTAACAGTCTGTCGCTGCTGACCCAGAATAACCTGAACAAATCCCAGTCCGCACTGGGCACCGCTATCGAGCGTCTGTCTTCTGGTCTGCGTATCAACAGCGCGAAAGACGATGCGGCAGGTCAGGCGATTGCTAACCGTTTCACCGCGAACATCAAAGGTCTGACTCAGGCTTCCCGTAACGCTAACGACGGTATCTCCATTGCGCAGACCACTGAAGGCGCGCTGAACGAAATCAACAACAACCTGCAGCGTGTGCGTGAACTGGCGGTTCAGTCTGCTAACAGCACCAACTCCCAGTCTGACCTCGACTCCATCCAGGCTGAAATCACCCAGCGCCTGAACGAAATCGACCGTGTATCCGGCCAGACTCAGTTCAACGGCGTGAAAGTCCTGGCGCAGGACAACACCCTGACCATCCAGGTTGGCGCCAACGACGGTGAAACTATCGATATCGATCTGAAGCAGATCAACTCTCAGACCCTGGGTCTGGACTCACTGAACGTGCAGAAAGCGTATGATGTGAAAGATACAGCAGTAACAACGAAAGCTTATGCCAATAATGGTACTACACTGGATGTATCGGGTCTTGATGATGCAGCTATTAAAGCGGCTACGGGTGGTACGAATGGTACGGCTTCTGTAACCGGTGGTGCGGTTAAATTTGACGCAGATAATAACAAGTACTTTGTTACTATTGGTGGCTTTACTGGTGCTGATGCCGCCAAAAATGGCGATTATGAAGTTAACGTTGCTACTGACGGTACAGTAACCCTTGCGGCTGGCGCAACTAAAACCACAATGCCTGCTGGTGCGACAACTAAAACAGAAGTACAGGAGTTAAAAGATACACCGGCAGTTGTTTCAGCAGATGCTAAAAATGCCTTAATTGCTGGCGGCGTTGACGCTACCGATGCTAATGGCGCTGAGTTGGTCAAAATGTCTTATACCGATAAAAATGGTAAGACAATTGAAGGCGGTTATGCGCTTAAAGCTGGCGATAAGTATTACGCCGCAGATTACGATGAAGCGACAGGAGCAATTAAAGCTAAAACCACAAGTTATACTGCTGCTGACGGCACTACCAAAACAGCGGCTAACCAACTGGGTGGCGTAGACGGTAAAACCGAAGTCGTTACTATCGACGGTAAAACCTACAATGCCAGCAAAGCCGCTGGTCATGATTTCAAAGCACAACCAGAGCTGGCGGAAGCAGCCGCTAAAACCACCGAAAACCCGCTGCAGAAAATTGATGCCGCGCTGGCGCAGGTGGATGCGCTGCGCTCTGATCTGGGTGCGGTACAAAACCGTTTCAACTCTGCTATCACCAACCTGGGCAATACCGTAAACAATCTGTCTGAAGCGCGTAGCCGTATCGAAGATTCCGACTACGCGACCGAAGTTTCCAACATGTCTCGCGCGCAGATTTTGCAGCAGGCCGGTACTTCCGTTCTGGCGCAGGCTAACCAGGTCCCGCAGAACGTGCTGAGCCTGTTAGCGGGTGCGAAACCGCTGTCTCTGCGCGATTGCTCTGTGGCAGGCTGGCTGCTGGGCAACCCTATGTGTGACGAATTCATTAACGTTCCGGAATGGTCTGATATTGCCGAAAAAGCTAACCCTGTCAACGATCTGTGTTACCCTGGTGACTTTAACGATTACGAAGAACTGAAGCACCTGCTGTCTCGTATCAATCACTTCGAGAAAATCCAGATCATCCCGAAATCCTCCTGGAGCTCCCACGAAGCTTCTCTGGGCGTATCCTCCGCGTGCCCGTACCAGGGCAAATCCTCTTTCTTTCGTAACGTTGTTTGGCTGATCAAGAAAAACTCCACCTACCCGACGATCAAGCGTAGCTATAATAACACCAACCAGGAAGACCTGCTGGTTCTGTGGGGCATCCACCATCCAAACGATGCTGCGGAACAGACCAAGCTGTACCAGAACCCGACCACCTACATCAGCGTGGGCACCTCTACGCTGAACCAGCGTCTGGTACCGCGTATCGCAACCCGCAGCAAGGTAAACGGTCAAAGCGGCCGCATGGAATTTTTCTGGACCATCCTGAAACCGAACGACGCAATCAACTTCGAATCTAACGGCAATTTCATCGCTCCGGAGTATGCGTACAAAATCGTAAAGAAAGGTGATAGCACTATCATGAAATCCGAGTAGTAA HA1-2mut VN SEQ ID NO: 72GGTGCGAAACCGCTGTCTCTGCGCGATTGCTCTGTGGCAGGCTGGCTGCTGGGCAACCCTATGTGTGACGAATTCATTAACGTTCCGGAATGGTCTGATATTGCCGAAAAAGCTAACCCTGTCAACGATCTGTGTTACCCTGGTGACTTTAACGATTACGAAGAACTGAAGCACCTGCTGTCTCGTATCAATCACTTCGAGAAAATCCAGATCATCCCGAAATCCTCCTGGAGCTCCCACGAAGCTTCTCTGGGCGTATCCTCCGCGTGCCCGTACCAGGGCAAATCCTCTTTCTTTCGTAACGTTGTTTGGCTGATCAAGAAAAACTCCACCTACCCGACGATCAAGCGTAGCTATAATAACACCAACCAGGAAGACCTGCTGGTTCTGTGGGGCATCCACCATCCAAACGATGCTGCGGAACAGACCAAGCTGTACCAGAACCCGACCACCTACATCAGCGTGGGCACCTCTACGCTGAACCAGCGTCTGGTACCGCGTATCGCAACCCGCAGCAAGGTAAACGGTCAAAGCGGCCGCATGGAATTTTTCTGGACCATCCTGAAACCGAACGACGCAATCAACTTCGAATCTAACGGCAATTTCATCGCTCCGGAGTATGCGTACAAAATCGTAAAGAAAGGTGATAGCACTATCATGAAATCCGAGTAGTAA STF2.HA1-1 IND SEQ ID NO: 73ATGGCACAAGTAATCAACACTAACAGTCTGTCGCTGCTGACCCAGAATAACCTGAACAAATCCCAGTCCGCACTGGGCACCGCTATCGAGCGTCTGTCTTCTGGTCTGCGTATCAACAGCGCGAAAGACGATGCGGCAGGTCAGGCGATTGCTAACCGTTTCACCGCGAACATCAAAGGTCTGACTCAGGCTTCCCGTAACGCTAACGACGGTATCTCCATTGCGCAGACCACTGAAGGCGCGCTGAACGAAATCAACAACAACCTGCAGCGTGTGCGTGAACTGGCGGTTCAGTCTGCTAACAGCACCAACTCCCAGTCTGACCTCGACTCCATCCAGGCTGAAATCACCCAGCGCCTGAACGAAATCGACCGTGTATCCGGCCAGACTCAGTTCAACGGCGTGAAAGTCCTGGCGCAGGACAACACCCTGACCATCCAGGTTGGCGCCAACGACGGTGAAACTATCGATATCGATCTGAAGCAGATCAACTCTCAGACCCTGGGTCTGGACTCACTGAACGTGCAGAAAGCGTATGATGTGAAAGATACAGCAGTAACAACGAAAGCTTATGCCAATAATGGTACTACACTGGATGTATCGGGTCTTGATGATGCAGCTATTAAAGCGGCTACGGGTGGTACGAATGGTACGGCTTCTGTAACCGGTGGTGCGGTTAAATTTGACGCAGATAATAACAAGTACTTTGTTACTATTGGTGGCTTTACTGGTGCTGATGCCGCCAAAAATGGCGATTATGAAGTTAACGTTGCTACTGACGGTACAGTAACCCTTGCGGCTGGCGCAACTAAAACCACAATGCCTGCTGGTGCGACAACTAAAACAGAAGTACAGGAGTTAAAAGATACACCGGCAGTTGTTTCAGCAGATGCTAAAAATGCCTTAATTGCTGGCGGCGTTGACGCTACCGATGCTAATGGCGCTGAGTTGGTCAAAATGTCTTATACCGATAAAAATGGTAAGACAATTGAAGGCGGTTATGCGCTTAAAGCTGGCGATAAGTATTACGCCGCAGATTACGATGAAGCGACAGGAGCAATTAAAGCTAAAACCACAAGTTATACTGCTGCTGACGGCACTACCAAAACAGCGGCTAACCAACTGGGTGGCGTAGACGGTAAAACCGAAGTCGTTACTATCGACGGTAAAACCTACAATGCCAGCAAAGCCGCTGGTCATGATTTCAAAGCACAACCAGAGCTGGCGGAAGCAGCCGCTAAAACCACCGAAAACCCGCTGCAGAAAATTGATGCCGCGCTGGCGCAGGTGGATGCGCTGCGCTCTGATCTGGGTGCGGTACAAAACCGTTTCAACTCTGCTATCACCAACCTGGGCAATACCGTAAACAATCTGTCTGAAGCGCGTAGCCGTATCGAAGATTCCGACTACGCGACCGAAGTTTCCAACATGTCTCGCGCGCAGATTTTGCAGCAGGCCGGTACTTCCGTTCTGGCGCAGGCTAACCAGGTCCCGCAGAACGTGCTGTCTCTGTTAGCGGAGAAAACGCATAACGGTAAACTGTGCGACCTGGACGGCGTGAAGCCGCTGATCCTGCGTGACTGTTCTGTTGCTGGCTGGCTGCTGGGCAACCCGATGTGCGATGAGTTCATCAACGTTCCGGAGTGGTCTTATATTGTTGAAAAAGCGAACCCGACCAATGACCTGTGCTACCCGGGCTCTTTTAATGACTACGAGGAGCTGAAACATCTGCTGTCTCGCATCAACCATTTCGAAAAAATTCAGATCATCCCGAAATCTTCCTGGAGCGATCACGAAGCTTCTTCCGGCGTATCTTCTGCATGCCCTTACCTGGGTTCCCCGTCTTTCTTCCGTAACGTGGTTTGGCTGATCAAGAAGAACTCTACGTACCCGACCATCAAGAAATCCTATAACAACACTAACCAGGAAGATCTGCTGGTGCTGTGGGGTATCCATCATCCAAACGATGCAGCCGAACAGACCCGCCTGTACCAGAATCCGACCACGTACATCTCTATCGGCACTTCTACCCTGAATCAACGCCTGGTGCCGAAAATCGCAACTCGCAGCAAAGTCAACGGCCAATCTGGTCGCATGGAGTTTTTCTGGACCATCCTGAAACCGAACGACGCGATTAACTTCGAAAGCAACGGCAACTTCATTGCACCGGAATATGCTTACAAGATCGTTAAAAAGGGTGATTCTGCGATCATGAAAAGCGAGCTGGAATACGGCAACTGCAACACTAAATGCCAGACCCCGATGGGTGCAATCAATTCCAGCATGCCATTTCACAACATCCACCCGCTGACCATCGGCGAGTGTCCAAAATACGTTAAA TAATAGFOR PRIMER SEQ ID NO: 74 AGTGGCTGAGCCTGTTAGCGGAGAAAACGCATAACGGTAAACTGTGHA0s IND SEQ ID NO: 75GCTGAGCCTGTTACGTGATCAAATCTGCATCGGTTACCACGCAAACAACTCCACTGAACAAGTGGATACGATCATGGAAAAGAACGTGACCGTGACCCACGCTCAAGATATCCTGGAGAAAACGCATAACGGTAAACTGTGCGACCTGGACGGCGTGAAGCCGCTGATCCTGCGTGACTGTTCTGTTGCTGGCTGGCTGCTGGGCAACCCGATGTGCGATGAGTTCATCAACGTTCCGGAGTGGTCTTATATTGTTGAAAAAGCGAACCCGACCAATGACCTGTGCTACCCGGGCTCTTTTAATGACTACGAGGAGCTGAAACATCTGCTGTCTCGCATCAACCATTTCGAAAAAATTCAGATCATCCCGAAATCTTCCTGGAGCGATCACGAAGCTTCTTCCGGCGTATCTTCTGCATGCCCTTACCTGGGTTCCCCGTCTTTCTTCCGTAACGTGGTTTGGCTGATCAAGAAGAACTCTACGTACCCGACCATCAAGAAATCCTATAACAACACTAACCAGGAAGATCTGCTGGTGCTGTGGGGTATCCATCATCCAAACGATGCAGCCGAACAGACCCGCCTGTACCAGAATCCGACCACGTACATCTCTATCGGCACTTCTACCCTGAATCAACGCCTGGTGCCGAAAATCGCAACTCGCAGCAAAGTCAACGGCCAATCTGGTCGCATGGAGTTTTTCTGGACCATCCTGAAACCGAACGACGCGATTAACTTCGAAAGCAACGGCAACTTCATTGCACCGGAATATGCTTACAAGATCGTTAAAAAGGGTGATTCTGCGATCATGAAAAGCGAGCTGGAATACGGCAACTGCAACACTAAATGCCAGACCCCGATGGGTGCAATCAATTCCAGCATGCCATTTCACAACATCCACCCGCTGACCATCGGCGAGTGTCCAAAATACGTTAAATCCAATCGTCTGGTGCTGGCAACGGGCCTGCGCAACTCTCCGCAACGTGAGTCTCGTCGCAAAAAGCGTGGCCTGTTTGGCGCAATTGCGGGCTTCATCGAGGGCGGCTGGCAGGGTATGGTTGATGGTTGGTACGGTTATCACCATTCCAACGAGCAGGGCTCTGGTTACGCTGCGGACAAAGAGTCCACGCAGAAAGCGATCGATGGCGTCACCAACAAAGTGAACTCTATCATCGACAAGATGAACACTCAGTTCGAGGCTGTGGGCCGTGAATTCAACAATCTGGAACGTCGCATCGAAAACCTGAACAAAAAGATGGAAGACGGTTTCCTGGACGTATGGACCTATAACGCAGAGCTGCTGGTTCTGATGGAAAACGAACGTACTCTGGACTTCCACGATTCTAACGTTAAAAACCTGTACGACAAAGTTCGTCTGCAGCTGCGCGATAATGCAAAAGAACTGGGTAACGGCTGCTTTGAATTTTACCACAAATGTGACAACGAATGCATGGAAAGCATCCGTAACGGTACCTACAATTACCCACAGTACTCCGAAGAAGCGCGTCTGAAACGTGAAGAAATCTCCGGTGTAAAACTGGAATCCATTGGCACGTATCAGATTCTGTCTATCTACTCCACGGTAGCGTCCTCTCTGGCGCTGGCAATTATGATGGCCGGCCTGTCTCTGTGGATGTGCTCTAACGGCTCTCTGCAGTGCCGCATCTGCATCTAATAGGC TGAGCSTF2.HA1-2 IND SEQ ID NO: 76ATGGCACAAGTAATCAACACTAACAGTCTGTCGCTGCTGACCCAGAATAACCTGAACAAATCCCAGTCCGCACTGGGCACCGCTATCGAGCGTCTGTCTTCTGGTCTGCGTATCAACAGCGCGAAAGACGATGCGGCAGGTCAGGCGATTGCTAACCGTTTCACCGCGAACATCAAAGGTCTGACTCAGGCTTCCCGTAACGCTAACGACGGTATCTCCATTGCGCAGACCACTGAAGGCGCGCTGAACGAAATCAACAACAACCTGCAGCGTGTGCGTGAACTGGCGGTTCAGTCTGCTAACAGCACCAACTCCCAGTCTGACCTCGACTCCATCCAGGCTGAAATCACCCAGCGCCTGAACGAAATCGACCGTGTATCCGGCCAGACTCAGTTCAACGGCGTGAAAGTCCTGGCGCAGGACAACACCCTGACCATCCAGGTTGGCGCCAACGACGGTGAAACTATCGATATCGATCTGAAGCAGATCAACTCTCAGACCCTGGGTCTGGACTCACTGAACGTGCAGAAAGCGTATGATGTGAAAGATACAGCAGTAACAACGAAAGCTTATGCCAATAATGGTACTACACTGGATGTATCGGGTCTTGATGATGCAGCTATTAAAGCGGCTACGGGTGGTACGAATGGTACGGCTTCTGTAACCGGTGGTGCGGTTAAATTTGACGCAGATAATAACAAGTACTTTGTTACTATTGGTGGCTTTACTGGTGCTGATGCCGCCAAAAATGGCGATTATGAAGTTAACGTTGCTACTGACGGTACAGTAACCCTTGCGGCTGGCGCAACTAAAACCACAATGCCTGCTGGTGCGACAACTAAAACAGAAGTACAGGAGTTAAAAGATACACCGGCAGTTGTTTCAGCAGATGCTAAAAATGCCTTAATTGCTGGCGGCGTTGACGCTACCGATGCTAATGGCGCTGAGTTGGTCAAAATGTCTTATACCGATAAAAATGGTAAGACAATTGAAGGCGGTTATGCGCTTAAAGCTGGCGATAAGTATTACGCCGCAGATTACGATGAAGCGACAGGAGCAATTAAAGCTAAAACCACAAGTTATACTGCTGCTGACGGCACTACCAAAACAGCGGCTAACCAACTGGGTGGCGTAGACGGTAAAACCGAAGTCGTTACTATCGACGGTAAAACCTACAATGCCAGCAAAGCCGCTGGTCATGATTTCAAAGCACAACCAGAGCTGGCGGAAGCAGCCGCTAAAACCACCGAAAACCCGCTGCAGAAAATTGATGCCGCGCTGGCGCAGGTGGATGCGCTGCGCTCTGATCTGGGTGCGGTACAAAACCGTTTCAACTCTGCTATCACCAACCTGGGCAATACCGTAAACAATCTGTCTGAAGCGCGTAGCCGTATCGAAGATTCCGACTACGCGACCGAAGTTTCCAACATGTCTCGCGCGCAGATTTTGCAGCAGGCCGGTACTTCCGTTCTGGCGCAGGCTAACCAGGTCCCGCAGAACGTGCTGAGCCTGTTAGCAGGTGTGAAACCGCTGATTCTGCGCGATTGTTCTGTTGCTGGCTGGCTGCTGGGCAACCCGATGTGCGATGAGTTCATCAACGTTCCGGAGTGGTCTTATATTGTTGAAAAAGCGAACCCGACCAATGACCTGTGCTACCCGGGCTCTTTTAATGACTACGAGGAGCTGAAACATCTGCTGTCTCGCATCAACCATTTCGAAAAAATTCAGATCATCCCGAAATCTTCCTGGAGCGATCACGAAGCTTCTTCCGGCGTATCTTCTGCATGCCCTTACCTGGGTTCCCCGTCTTTCTTCCGTAACGTGGTTTGGCTGATCAAGAAGAACTCTACGTACCCGACCATCAAGAAATCCTATAACAACACTAACCAGGAAGATCTGCTGGTGCTGTGGGGTATCCATCATCCAAACGATGCAGCCGAACAGACCCGCCTGTACCAGAATCCGACCACGTACATCTCTATCGGCACTTCTACCCTGAATCAACGCCTGGTGCCGAAAATCGCAACTCGCAGCAAAGTCAACGGCCAATCTGGTCGCATGGAGTTTTTCTGGACCATCCTGAAACCGAACGACGCGATTAACTTCGAAAGCAACGGCAACTTCATTGCACCGGAATATGCTTACAAGATCGTTAAAAAGGGTGATTCTGCGATCATGAAAAGCGAGTAATAG REV PRIMER SEQ ID NO: 77AGTCGCTCAGCCTATTACTCGCTTTTCATGATCGCAGAATCAC STF2.HA1-2mut INDSEQ ID NO: 78ATGGCACAAGTAATCAACACTAACAGTCTGTCGCTGCTGACCCAGAATAACCTGAACAAATCCCAGTCCGCACTGGGCACCGCTATCGAGCGTCTGTCTTCTGGTCTGCGTATCAACAGCGCGAAAGACGATGCGGCAGGTCAGGCGATTGCTAACCGTTTCACCGCGAACATCAAAGGTCTGACTCAGGCTTCCCGTAACGCTAACGACGGTATCTCCATTGCGCAGACCACTGAAGGCGCGCTGAACGAAATCAACAACAACCTGCAGCGTGTGCGTGAACTGGCGGTTCAGTCTGCTAACAGCACCAACTCCCAGTCTGACCTCGACTCCATCCAGGCTGAAATCACCCAGCGCCTGAACGAAATCGACCGTGTATCCGGCCAGACTCAGTTCAACGGCGTGAAAGTCCTGGCGCAGGACAACACCCTGACCATCCAGGTTGGCGCCAACGACGGTGAAACTATCGATATCGATCTGAAGCAGATCAACTCTCAGACCCTGGGTCTGGACTCACTGAACGTGCAGAAAGCGTATGATGTGAAAGATACAGCAGTAACAACGAAAGCTTATGCCAATAATGGTACTACACTGGATGTATCGGGTCTTGATGATGCAGCTATTAAAGCGGCTACGGGTGGTACGAATGGTACGGCTTCTGTAACCGGTGGTGCGGTTAAATTTGACGCAGATAATAACAAGTACTTTGTTACTATTGGTGGCTTTACTGGTGCTGATGCCGCCAAAAATGGCGATTATGAAGTTAACGTTGCTACTGACGGTACAGTAACCCTTGCGGCTGGCGCAACTAAAACCACAATGCCTGCTGGTGCGACAACTAAAACAGAAGTACAGGAGTTAAAAGATACACCGGCAGTTGTTTCAGCAGATGCTAAAAATGCCTTAATTGCTGGCGGCGTTGACGCTACCGATGCTAATGGCGCTGAGTTGGTCAAAATGTCTTATACCGATAAAAATGGTAAGACAATTGAAGGCGGTTATGCGCTTAAAGCTGGCGATAAGTATTACGCCGCAGATTACGATGAAGCGACAGGAGCAATTAAAGCTAAAACCACAAGTTATACTGCTGCTGACGGCACTACCAAAACAGCGGCTAACCAACTGGGTGGCGTAGACGGTAAAACCGAAGTCGTTACTATCGACGGTAAAACCTACAATGCCAGCAAAGCCGCTGGTCATGATTTCAAAGCACAACCAGAGCTGGCGGAAGCAGCCGCTAAAACCACCGAAAACCCGCTGCAGAAAATTGATGCCGCGCTGGCGCAGGTGGATGCGCTGCGCTCTGATCTGGGTGCGGTACAAAACCGTTTCAACTCTGCTATCACCAACCTGGGCAATACCGTAAACAATCTGTCTGAAGCGCGTAGCCGTATCGAAGATTCCGACTACGCGACCGAAGTTTCCAACATGTCTCGCGCGCAGATTTTGCAGCAGGCCGGTACTTCCGTTCTGGCGCAGGCTAACCAGGTCCCGCAGAACGTGCTGTCTCTGTTAGCGGGCGCAAAGCCGCTGTCTCTGCGTGACTGTTCTGTTGCTGGCTGGCTGCTGGGCAACCCGATGTGCGATGAGTTCATCAACGTTCCGGAGTGGTCTGATATTGCGGAAAAAGCGAACCCGACCAATGACCTGTGCTACCCGGGCTCTTTTAATGACTACGAGGAGCTGAAACATCTGCTGTCTCGCATCAACCATTTCGAAAAAATTCAGATCATCCCGAAATCTTCCTGGAGCGATCACGAAGCTTCTTCCGGCGTATCTTCTGCATGCCCTTACCTGGGTTCCCCGTCTTTCTTCCGTAACGTGGTTTGGCTGATCAAGAAGAACTCTACGTACCCGACCATCAAGAAATCCTATAACAACACTAACCAGGAAGATCTGCTGGTGCTGTGGGGTATCCATCATCCAAACGATGCAGCCGAACAGACCCGCCTGTACCAGAATCCGACCACGTACATCTCTATCGGCACTTCTACCCTGAATCAACGCCTGGTGCCGAAAATCGCAACTCGCAGCAAAGTCAACGGCCAATCTGGTCGCATGGAGTTTTTCTGGACCATCCTGAAACCGAACGACGCGATTAACTTCGAAAGCAACGGCAACTTCATTGCACCGGAATATGCTTACAAGATCGTTAAAAAGGGTGATTCTGCGATCATGAAAAGCGAGTAATAG HA1-2mut IND SEQ ID 79GGCGCAAAGCCGCTGTCTCTGCGTGACTGTTCTGTTGCTGGCTGGCTGCTGGGCAACCCGATGTGCGATGAGTTCATCAACGTTCCGGAGTGGTCTGATATTGCGGAAAAAGCGAACCCGACCAATGACCTGTGCTACCCGGGCTCTTTTAATGACTACGAGGAGCTGAAACATCTGCTGTCTCGCATCAACCATTTCGAAAAAATTCAGATCATCCCGAAATCTTCCTGGAGCGATCACGAAGCTTCTTCCGGCGTATCTTCTGCATGCCCTTACCTGGGTTCCCCGTCTTTCTTCCGTAACGTGGTTTGGCTGATCAAGAAGAACTCTACGTACCCGACCATCAAGAAATCCTATAACAACACTAACCAGGAAGATCTGCTGGTGCTGTGGGGTATCCATCATCCAAACGATGCAGCCGAACAGACCCGCCTGTACCAGAATCCGACCACGTACATCTCTATCGGCACTTCTACCCTGAATCAACGCCTGGTGCCGAAAATCGCAACTCGCAGCAAAGTCAACGGCCAATCTGGTCGCATGGAGTTTTTCTGGACCATCCTGAAACCGAACGACGCGATTAACTTCGAAAGCAACGGCAACTTCATTGCACCGGAATATGCTTACAAGATCGTTAAAAAGGGTGATTCTGCGATCATGAAAAGCGAGTAATAGGCTGAGC STF2.HA1-1 NC SEQ ID NO: 80ATGGCACAAGTAATCAACACTAACAGTCTGTCGCTGCTGACCCAGAATAACCTGAACAAATCCCAGTCCGCACTGGGCACCGCTATCGAGCGTCTGTCTTCTGGTCTGCGTATCAACAGCGCGAAAGACGATGCGGCAGGTCAGGCGATTGCTAACCGTTTCACCGCGAACATCAAAGGTCTGACTCAGGCTTCCCGTAACGCTAACGACGGTATCTCCATTGCGCAGACCACTGAAGGCGCGCTGAACGAAATCAACAACAACCTGCAGCGTGTGCGTGAACTGGCGGTTCAGTCTGCTAACAGCACCAACTCCCAGTCTGACCTCGACTCCATCCAGGCTGAAATCACCCAGCGCCTGAACGAAATCGACCGTGTATCCGGCCAGACTCAGTTCAACGGCGTGAAAGTCCTGGCGCAGGACAACACCCTGACCATCCAGGTTGGCGCCAACGACGGTGAAACTATCGATATCGATCTGAAGCAGATCAACTCTCAGACCCTGGGTCTGGACTCACTGAACGTGCAGAAAGCGTATGATGTGAAAGATACAGCAGTAACAACGAAAGCTTATGCCAATAATGGTACTACACTGGATGTATCGGGTCTTGATGATGCAGCTATTAAAGCGGCTACGGGTGGTACGAATGGTACGGCTTCTGTAACCGGTGGTGCGGTTAAATTTGACGCAGATAATAACAAGTACTTTGTTACTATTGGTGGCTTTACTGGTGCTGATGCCGCCAAAAATGGCGATTATGAAGTTAACGTTGCTACTGACGGTACAGTAACCCTTGCGGCTGGCGCAACTAAAACCACAATGCCTGCTGGTGCGACAACTAAAACAGAAGTACAGGAGTTAAAAGATACACCGGCAGTTGTTTCAGCAGATGCTAAAAATGCCTTAATTGCTGGCGGCGTTGACGCTACCGATGCTAATGGCGCTGAGTTGGTCAAAATGTCTTATACCGATAAAAATGGTAAGACAATTGAAGGCGGTTATGCGCTTAAAGCTGGCGATAAGTATTACGCCGCAGATTACGATGAAGCGACAGGAGCAATTAAAGCTAAAACCACAAGTTATACTGCTGCTGACGGCACTACCAAAACAGCGGCTAACCAACTGGGTGGCGTAGACGGTAAAACCGAAGTCGTTACTATCGACGGTAAAACCTACAATGCCAGCAAAGCCGCTGGTCATGATTTCAAAGCACAACCAGAGCTGGCGGAAGCAGCCGCTAAAACCACCGAAAACCCGCTGCAGAAAATTGATGCCGCGCTGGCGCAGGTGGATGCGCTGCGCTCTGATCTGGGTGCGGTACAAAACCGTTTCAACTCTGCTATCACCAACCTGGGCAATACCGTAAACAATCTGTCTGAAGCGCGTAGCCGTATCGAAGATTCCGACTACGCGACCGAAGTTTCCAACATGTCTCGCGCGCAGATTTTGCAGCAGGCCGGTACTTCCGTTCTGGCGCAGGCTAACCAGGTCCCGCAGAACGTGCTGAGCCTGTTAGCGTCCCACAACGGTAAACTGTGTCTGCTGAAAGGCATCGCACCGCTGCAGCTGGGTAACTGTAGCGTTGCAGGTTGGATCCTGGGTAACCCGGAGTGCGAACTGCTGATTTCCAAGGAGAGCTGGTCTTACATTGTCGAAACGCCGAATCCGGAAAACGGCACTTGTTATCCGGGTTACTTCGCCGATTACGAAGAACTGCGTGAACAACTGTCTTCCGTGAGCTCTTTCGAACGTTTCGAGATCTTCCCGAAAGAGAGCAGCTGGCCAAACCACACTGTTACCGGTGTGTCTGCGAGCTGTTCTCACAACGGCAAGTCTTCTTTCTACCGCAACCTGCTGTGGCTGACGGGTAAGAATGGCCTGTATCCGAACCTGTCTAAATCTTACGTAAACAACAAAGAGAAAGAGGTGCTGGTCCTGTGGGGCGTACACCATCCACCAAATATCGGCAACCAGCGCGCCCTGTACCACACCGAAAACGCTTATGTGTCCGTGGTGAGCTCCCATTACAGCCGTCGTTTTACTCCGGAGATTGCCAAACGTCCGAAAGTTCGTGATCAGGAAGGCCGTATTAACTACTACTGGACTCTGCTGGAGCCGGGCGATACCATCATTTTCGAGGCAAACGGCAACCTGATTGCGCCATGGTACGCGTTCGCCCTGAGCCGTGGTTTTGGCTCCGGTATTATCACCTCTAACGCGCCAATGGACGAATGCGACGCGAAATGCCAAACGCCGCAGGGCGCAATCAACAGCAGCCTGCCGTTCCAGAACGTTCACCCGGTTACCATCGGCGAATGCCCTAAATACGTGCGCTAATAG FOR PRIMERSEQ ID NO: 81AACCAGGTCCCGCAGAACGTGCTGAGCCTGTTAGCGTCCCACAACGGTAAACTGTGTCTGCTGAAAGGCATCGCACCGCTGCAG REV PRIMER SEQ ID NO: 82GAACAGCTCAGTTCCTACGAGCTCAGCCTATTAGCGCACGTATTTAGGGCATTCGCCGATGGTAACCGGGTGAACGTT HA0s NC SEQ ID NO: 83GACACGATCTGTATTGGTTATCATGCAAACAACTCTACTGACACTGTAGATACTGTGCTGGAAAAGAACGTAACCGTTACCCACAGCGTTAACCTGCTGGAAGATTCCCACAACGGTAAACTGTGTCTGCTGAAAGGCATCGCACCGCTGCAGCTGGGTAACTGTAGCGTTGCAGGTTGGATCCTGGGTAACCCGGAGTGCGAACTGCTGATTTCCAAGGAGAGCTGGTCTTACATTGTCGAAACGCCGAATCCGGAAAACGGCACTTGTTATCCGGGTTACTTCGCCGATTACGAAGAACTGCGTGAACAACTGTCTTCCGTGAGCTCTTTCGAACGTTTCGAGATCTTCCCGAAAGAGAGCAGCTGGCCAAACCACACTGTTACCGGTGTGTCTGCGAGCTGTTCTCACAACGGCAAGTCTTCTTTCTACCGCAACCTGCTGTGGCTGACGGGTAAGAATGGCCTGTATCCGAACCTGTCTAAATCTTACGTAAACAACAAAGAGAAAGAGGTGCTGGTCCTGTGGGGCGTACACCATCCACCAAATATCGGCAACCAGCGCGCCCTGTACCACACCGAAAACGCTTATGTGTCCGTGGTGAGCTCCCATTACAGCCGTCGTTTTACTCCGGAGATTGCCAAACGTCCGAAAGTTCGTGATCAGGAAGGCCGTATTAACTACTACTGGACTCTGCTGGAGCCGGGCGATACCATCATTTTCGAGGCAAACGGCAACCTGATTGCGCCATGGTACGCGTTCGCCCTGAGCCGTGGTTTTGGCTCCGGTATTATCACCTCTAACGCGCCAATGGACGAATGCGACGCGAAATGCCAAACGCCGCAGGGCGCAATCAACAGCAGCCTGCCGTTCCAGAACGTTCACCCGGTTACCATCGGCGAATGCCCTAAATACGTGCGCTCCGCCAAACTGCGCATGGTTACTGGTCTGCGTAACATCCCGAGCATTCAGTCTCGCGGTCTGTTCGGTGCGATCGCGGGCTTCATTGAAGGCGGTTGGACCGGTATGGTGGATGGTTGGTACGGCTACCATCACCAGAACGAACAGGGTAGCGGTTACGCTGCCGACCAGAAATCCACCCAGAACGCTATTAACGGTATCACCAACAAAGTTAACAGCGTAATTGAGAAGATGAACACGCAGTTCACCGCCGTAGGTAAGGAATTCAACAAGCTGGAACGTCGCATGGAAAACCTGAACAAAAAGGTGGACGACGGCTTTCTGGACATCTGGACCTACAACGCTGAACTGCTGGTGCTGCTGGAAAACGAACGTACCCTGGATTTCCACGACTCTAATGTTAAAAACCTGTACGAAAAGGTCAAGTCTCAACTGAAAAACAATGCGAAGGAAATCGGCAACGGCTGTTTCGAATTCTACCATAAATGCAACAACGAATGCATGGAATCCGTTAAAAACGGTACCTATGACTACCCTAAATACTCCGAAGAAAGCAAACTGAACCGCGAGAAAATCGATGGTGTAAAACTGGAATCTATGGGTGTTTACCAGATCCTGGCGATCTACTCCACGGTAGCCAGCAGCCTGGTTCTGCTGGTTAGCCTGGGTGCAATTAGCTTCTGGATGTGCTCTAACGGCAGCCTGCAATGCCGCATCTGTAATAGGCTGAGC STF2HA1-2 NC SEQ ID NO: 84ATGGCACAAGTAATCAACACTAACAGTCTGTCGCTGCTGACCCAGAATAACCTGAACAAATCCCAGTCCGCACTGGGCACCGCTATCGAGCGTCTGTCTTCTGGTCTGCGTATCAACAGCGCGAAAGACGATGCGGCAGGTCAGGCGATTGCTAACCGTTTCACCGCGAACATCAAAGGTCTGACTCAGGCTTCCCGTAACGCTAACGACGGTATCTCCATTGCGCAGACCACTGAAGGCGCGCTGAACGAAATCAACAACAACCTGCAGCGTGTGCGTGAACTGGCGGTTCAGTCTGCTAACAGCACCAACTCCCAGTCTGACCTCGACTCCATCCAGGCTGAAATCACCCAGCGCCTGAACGAAATCGACCGTGTATCCGGCCAGACTCAGTTCAACGGCGTGAAAGTCCTGGCGCAGGACAACACCCTGACCATCCAGGTTGGCGCCAACGACGGTGAAACTATCGATATCGATCTGAAGCAGATCAACTCTCAGACCCTGGGTCTGGACTCACTGAACGTGCAGAAAGCGTATGATGTGAAAGATACAGCAGTAACAACGAAAGCTTATGCCAATAATGGTACTACACTGGATGTATCGGGTCTTGATGATGCAGCTATTAAAGCGGCTACGGGTGGTACGAATGGTACGGCTTCTGTAACCGGTGGTGCGGTTAAATTTGACGCAGATAATAACAAGTACTTTGTTACTATTGGTGGCTTTACTGGTGCTGATGCCGCCAAAAATGGCGATTATGAAGTTAACGTTGCTACTGACGGTACAGTAACCCTTGCGGCTGGCGCAACTAAAACCACAATGCCTGCTGGTGCGACAACTAAAACAGAAGTACAGGAGTTAAAAGATACACCGGCAGTTGTTTCAGCAGATGCTAAAAATGCCTTAATTGCTGGCGGCGTTGACGCTACCGATGCTAATGGCGCTGAGTTGGTCAAAATGTCTTATACCGATAAAAATGGTAAGACAATTGAAGGCGGTTATGCGCTTAAAGCTGGCGATAAGTATTACGCCGCAGATTACGATGAAGCGACAGGAGCAATTAAAGCTAAAACCACAAGTTATACTGCTGCTGACGGCACTACCAAAACAGCGGCTAACCAACTGGGTGGCGTAGACGGTAAAACCGAAGTCGTTACTATCGACGGTAAAACCTACAATGCCAGCAAAGCCGCTGGTCATGATTTCAAAGCACAACCAGAGCTGGCGGAAGCAGCCGCTAAAACCACCGAAAACCCGCTGCAGAAAATTGATGCCGCGCTGGCGCAGGTGGATGCGCTGCGCTCTGATCTGGGTGCGGTACAAAACCGTTTCAACTCTGCTATCACCAACCTGGGCAATACCGTAAACAATCTGTCTGAAGCGCGTAGCCGTATCGAAGATTCCGACTACGCGACCGAAGTTTCCAACATGTCTCGCGCGCAGATTTTGCAGCAGGCCGGTACTTCCGTTCTGGCGCAGGCTAACCAGGTCCCGCAGAACGTGCTGAGCCTGTTAGCGAAAGGCATCGCACCGCTGCAGCTGGGTAACTGTAGCGTTGCAGGTTGGATCCTGGGTAACCCGGAGTGCGAACTGCTGATTTCCAAGGAGAGCTGGTCTTACATTGTCGAAACGCCGAATCCGGAAAACGGCACTTGTTATCCGGGTTACTTCGCCGATTACGAAGAACTGCGTGAACAACTGTCTTCCGTGAGCTCTTTCGAACGTTTCGAGATCTTCCCGAAAGAGAGCAGCTGGCCAAACCACACTGTTACCGGTGTGTCTGCGAGCTGTTCTCACAACGGCAAGTCTTCTTTCTACCGCAACCTGCTGTGGCTGACGGGTAAGAATGGCCTGTATCCGAACCTGTCTAAATCTTACGTAAACAACAAAGAGAAAGAGGTGCTGGTCCTGTGGGGCGTACACCATCCACCAAATATCGGCAACCAGCGCGCCCTGTACCACACCGAAAACGCTTATGTGTCCGTGGTGAGCTCCCATTACAGCCGTCGTTTTACTCCGGAGATTGCCAAACGTCCGAAAGTTCGTGATCAGGAAGGCCGTATTAACTACTACTGGACTCTGCTGGAGCCGGGCGATACCATCATTTTCGAGGCAAACGGCAACCTGATTGCGCCATGGTACGCGTTCGCCCTGAGCCGTGGTTTTGGCTCCGGTATTATCACCTCTTAATAA FOR PRIMER SEQ ID NO: 85AACCAGGTCCCGCAGAACGTGCTGAGCCTGTTAGCGAAAGGCATCGCACCGCTGCAGCTGGGTAACTGTAGCGTTGCAGGTTGG REV PRIMER SEQ ID NO: 86GAACAGCTCAGTTCCTACGAGCTCAGCCTATTAAGAGGTGATAATACCGGAGCCAAAACCACGGCTCAGGGCGAACGCGTACCA STF2.HA1-2mut NC SEQ ID NO: 87ATGGCACAAGTAATCAACACTAACAGTCTGTCGCTGCTGACCCAGAATAACCTGAACAAATCCCAGTCCGCACTGGGCACCGCTATCGAGCGTCTGTCTTCTGGTCTGCGTATCAACAGCGCGAAAGACGATGCGGCAGGTCAGGCGATTGCTAACCGTTTCACCGCGAACATCAAAGGTCTGACTCAGGCTTCCCGTAACGCTAACGACGGTATCTCCATTGCGCAGACCACTGAAGGCGCGCTGAACGAAATCAACAACAACCTGCAGCGTGTGCGTGAACTGGCGGTTCAGTCTGCTAACAGCACCAACTCCCAGTCTGACCTCGACTCCATCCAGGCTGAAATCACCCAGCGCCTGAACGAAATCGACCGTGTATCCGGCCAGACTCAGTTCAACGGCGTGAAAGTCCTGGCGCAGGACAACACCCTGACCATCCAGGTTGGCGCCAACGACGGTGAAACTATCGATATCGATCTGAAGCAGATCAACTCTCAGACCCTGGGTCTGGACTCACTGAACGTGCAGAAAGCGTATGATGTGAAAGATACAGCAGTAACAACGAAAGCTTATGCCAATAATGGTACTACACTGGATGTATCGGGTCTTGATGATGCAGCTATTAAAGCGGCTACGGGTGGTACGAATGGTACGGCTTCTGTAACCGGTGGTGCGGTTAAATTTGACGCAGATAATAACAAGTACTTTGTTACTATTGGTGGCTTTACTGGTGCTGATGCCGCCAAAAATGGCGATTATGAAGTTAACGTTGCTACTGACGGTACAGTAACCCTTGCGGCTGGCGCAACTAAAACCACAATGCCTGCTGGTGCGACAACTAAAACAGAAGTACAGGAGTTAAAAGATACACCGGCAGTTGTTTCAGCAGATGCTAAAAATGCCTTAATTGCTGGCGGCGTTGACGCTACCGATGCTAATGGCGCTGAGTTGGTCAAAATGTCTTATACCGATAAAAATGGTAAGACAATTGAAGGCGGTTATGCGCTTAAAGCTGGCGATAAGTATTACGCCGCAGATTACGATGAAGCGACAGGAGCAATTAAAGCTAAAACCACAAGTTATACTGCTGCTGACGGCACTACCAAAACAGCGGCTAACCAACTGGGTGGCGTAGACGGTAAAACCGAAGTCGTTACTATCGACGGTAAAACCTACAATGCCAGCAAAGCCGCTGGTCATGATTTCAAAGCACAACCAGAGCTGGCGGAAGCAGCCGCTAAAACCACCGAAAACCCGCTGCAGAAAATTGATGCCGCGCTGGCGCAGGTGGATGCGCTGCGCTCTGATCTGGGTGCGGTACAAAACCGTTTCAACTCTGCTATCACCAACCTGGGCAATACCGTAAACAATCTGTCTGAAGCGCGTAGCCGTATCGAAGATTCCGACTACGCGACCGAAGTTTCCAACATGTCTCGCGCGCAGATTTTGCAGCAGGCCGGTACTTCCGTTCTGGCGCAGGCTAACCAGGTCCCGCAGAACGTGCTGAGCCTGTTAGCGAAAGGCGCGGCACCGCTGCAGCTGGGTAACTGTAGCGTTGCAGGTTGGATCCTGGGTAACCCGGAGTGCGAACTGCTGATTTCCAAGGAGAGCTGGTCTGATATTGCAGAAACGCCGAATCCGGAAAACGGCACTTGTTATCCGGGTTACTTCGCCGATTACGAAGAACTGCGTGAACAACTGTCTTCCGTGAGCTCTTTCGAACGTTTCGAGATCTTCCCGAAAGAGAGCAGCTGGCCAAACCACACTGTTACCGGTGTGTCTGCGAGCTGTTCTCACAACGGCAAGTCTTCTTTCTACCGCAACCTGCTGTGGCTGACGGGTAAGAATGGCCTGTATCCGAACCTGTCTAAATCTTACGTAAACAACAAAGAGAAAGAGGTGCTGGTCCTGTGGGGCGTACACCATCCACCAAATATCGGCAACCAGCGCGCCCTGTACCACACCGAAAACGCTTATGTGTCCGTGGTGAGCTCCCATTACAGCCGTCGTTTTACTCCGGAGATTGCCAAACGTCCGAAAGTTCGTGATCAGGAAGGCCGTATTAACTACTACTGGACTCTGCTGGAGCCGGGCGATACCATCATTTTCGAGGCAAACGGCAACCTGATTGCGCCATGGTACGCGTTCGCCCTGAGCCGTGGTTTTGGCTCCGGTATTATCACCTCTTAATAG HA1-2mut NC SEQ ID NO: 88GCTGAGCCTGTTAGCGAAAGGCGCGGCACCGCTGCAGCTGGGTAACTGTAGCGTTGCAGGTTGGATCCTGGGTAACCCGGAGTGCGAACTGCTGATTTCCAAGGAGAGCTGGTCTGATATTGCAGAAACGCCGAATCCGGAAAACGGCACTTGTTATCCGGGTTACTTCGCCGATTACGAAGAACTGCGTGAACAACTGTCTTCCGTGAGCTCTTTCGAACGTTTCGAGATCTTCCCGAAAGAGAGCAGCTGGCCAAACCACACTGTTACCGGTGTGTCTGCGAGCTGTTCTCACAACGGCAAGTCTTCTTTCTACCGCAACCTGCTGTGGCTGACGGGTAAGAATGGCCTGTATCCGAACCTGTCTAAATCTTACGTAAACAACAAAGAGAAAGAGGTGCTGGTCCTGTGGGGCGTACACCATCCACCAAATATCGGCAACCAGCGCGCCCTGTACCACACCGAAAACGCTTATGTGTCCGTGGTGAGCTCCCATTACAGCCGTCGTTTTACTCCGGAGATTGCCAAACGTCCGAAAGTTCGTGATCAGGAAGGCCGTATTAACTACTACTGGACTCTGCTGGAGCCGGGCGATACCATCATTTTCGAGGCAAACGGCAACCTGATTGCGCCATGGTACGCGTTCGCCCTGAGCCGTGGTTTTGGCTCCGGTATTATCACCTCTTAATAGGCTGAGC STF2.HA1-1his (PR8) SEQ ID NO: 89MAQVINTNSLSLLTQNNLNKSQSALGTAIERLSSGLRINSAKDDAAGQAIANRFTANIKGLTQASRNANDGISIAQTTEGALNEINNNLQRVRELAVQSANSTNSQSDLDSIQAEITQRLNEIDRVSGQTQFNGVKVLAQDNTLTIQVGANDGETIDIDLKQINSQTLGLDSLNVQKAYDVKDTAVTTKAYANNGTTLDVSGLDDAAIKAATGGTNGTASVTGGAVKFDADNNKYFVTIGGFTGADAAKNGDYEVNVATDGTVTLAAGATKTTMPAGATTKTEVQELKDTPAVVSADAKNALIAGGVDATDANGAELVKMSYTDKNGKTIEGGYALKAGDKYYAADYDEATGAIKAKTTSYTAADGTTKTAANQLGGVDGKTEVVTIDGKTYNASKAAGHDFKAQPELAEAAAKTTENPLQKIDAALAQVDALRSDLGAVQNRFNSAITNLGNTVNNLSEARSRIEDSDYATEVSNMSRAQILQQAGTSVLAQANQVPQNVLSLLASHNGKLCRLKGIAPLQLGKCNIAGWLLGNPECDPLLPVRSWSYIVETPNSENGICYPGDFIDYEELREQLSSVSSFERFEIFPKESSWPNHNTNGVTAACSHEGKSSFYRNLLWLTEKEGSYPKLKNSYVNKKGKEVLVLWGIHHPPNSKEQQNLYQNENAYVSVVTSNYNRRFTPEIAERPKVRDQAGRMNYYWTLLKPGDTIIFEANGNLIAPMYAFALSRGFGSGIITSNASMHECNTKCQTPLGAINSSLPYQNIHPVTIGECPKYVRHHHHHH STF2.HA1-2(PR8) SEQ ID NO: 90MAQVINTNSLSLLTQNNLNKSQSALGTAIERLSSGLRINSAKDDAAGQAIANRFTANIKGLTQASRNANDGISIAQTTEGALNEINNNLQRVRELAVQSANSTNSQSDLDSIQAEITQRLNEIDRVSGQTQFNGVKVLAQDNTLTIQVGANDGETIDIDLKQINSQTLGLDSLNVQKAYDVKDTAVTTKAYANNGTTLDVSGLDDAAIKAATGGTNGTASVTGGAVKFDADNNKYFVTIGGFTGADAAKNGDYEVNVATDGTVTLAAGATKTTMPAGATTKTEVQELKDTPAVVSADAKNALIAGGVDATDANGAELVKMSYTDKNGKTIEGGYALKAGDKYYAADYDEATGAIKAKTTSYTAADGTTKTAANQLGGVDGKTEVVTIDGKTYNASKAAGHDFKAQPELAEAAAKTTENPLQKIDAALAQVDALRSDLGAVQNRFNSAITNLGNTVNNLSEARSRIEDSDYATEVSNMSRAQILQQAGTSVLAQANQVPQNVLSLLAKGIAPLQLGKCNIAGWLLGNPECDPLLPVRSWSYIVETPNSENGICYPGDFIDYEELREQLSSVSSFERFEIFPKESSWPNHNTNGVTAACSHEGKSSFYRNLLWLTEKEGSYPKLKNSYVNKKGKEVLVLWGIHHPPNSKEQQNLYQNENAYVSVVTSNYNRRFTPEIAERPKVRDQAGRMNYYWTLLKPGDTIIFEANGNLIAPMYAFALSRGFGSGIITS STF2.HA1-2mut(PR8) SEQ ID NO: 91MAQVINTNSLSLLTQNNLNKSQSALGTAIERLSSGLRINSAKDDAAGQAIANRFTANIKGLTQASRNANDGISIAQTTEGALNEINNNLQRVRELAVQSANSTNSQSDLDSIQAEITQRLNEIDRVSGQTQFNGVKVLAQDNTLTIQVGANDGETIDIDLKQINSQTLGLDSLNVQKAYDVKDTAVTTKAYANNGTTLDVSGLDDAAIKAATGGTNGTASVTGGAVKFDADNNKYFVTIGGFTGADAAKNGDYEVNVATDGTVTLAAGATKTTMPAGATTKTEVQELKDTPAVVSADAKNALIAGGVDATDANGAELVKMSYTDKNGKTIEGGYALKAGDKYYAADYDEATGAIKAKTTSYTAADGTTKTAANQLGGVDGKTEVVTIDGKTYNASKAAGHDFKAQPELAEAAAKTTENPLQKIDAALAQVDALRSDLGAVQNRFNSAITNLGNTVNNLSEARSRIEDSDYATEVSNMSRAQILQQAGTSVLAQANQVPQNVLSLLAKGAAPLQLGKCNIAGWLLGNPECDPLLPVRSWSDIAETPNSENGICYPGDFIDYEELREQLSSVSSFERFEIFPKESSWPNHNTNGVTAACSHEGKSSFYRNLLWLTEKEGSYPKLKNSYVNKKGKEVLVLWGIHHPPNSKEQQNLYQNENAYVSVVTSNYNRRFTPEIAERPKVRDQAGRMNYYWTLLKPGDTIIFEANGNLIAPMYAFALSRGFGSGIITS STF2.HA1-3(PR8) SEQ ID NO: 92MAQVINTNSLSLLTQNNLNKSQSALGTAIERLSSGLRINSAKDDAAGQAIANRFTANIKGLTQASRNANDGISIAQTTEGALNEINNNLQRVRELAVQSANSTNSQSDLDSIQAEITQRLNEIDRVSGQTQFNGVKVLAQDNTLTIQVGANDGETIDIDLKQINSQTLGLDSLNVQKAYDVKDTAVTTKAYANNGTTLDVSGLDDAAIKAATGGTNGTASVTGGAVKFDADNNKYFVTIGGFTGADAAKNGDYEVNVATDGTVTLAAGATKTTMPAGATTKTEVQELKDTPAVVSADAKNALIAGGVDATDANGAELVKMSYTDKNGKTIEGGYALKAGDKYYAADYDEATGAIKAKTTSYTAADGTTKTAANQLGGVDGKTEVVTIDGKTYNASKAAGHDFKAQPELAEAAAKTTENPLQKIDAALAQVDALRSDLGAVQNRFNSAITNLGNTVNNLSEARSRIEDSDYATEVSNMSRAQILQQAGTSVLAQANQVPQNVLSLLANSENGICYPGDFIDYEELREQLSSVSSFERFEIFPKESSWPNHNTNGVTAACSHEGKSSFYRNLLWLTEKEGSYPKLKNSYVNKKGKEVLVLWGIHHPPNSKEQQNLYQNENAYVSVVTSNYNRRFTPEIAERPKVRDQAGRMNYYWTLLKPGDTIIFEANGNLIAPMYAFALSRG HA1-2his(PR8) SEQ ID NO: 93KGIAPLQLGKCNIAGWLLGNPECDPLLPVRSWSYIVETPNSENGICYPGDFIDYEELREQLSSVSSFERFEIFPKESSWPNHNTNGVTAACSHEGKSSFYRNLLWLTEKEGSYPKLKNSYVNKKGKEVLVLWGIHHPPNSKEQQNLYQNENAYVSVVTSNYNRRFTPEIAERPKVRDQAGRMNYYWTLLKPGDTIIFEANGNLIAPMYAFALSRGFGSGIITSHHHHHH STF2.4×M2e SEQ ID NO: 94MAQVINTNSLSLLTQNNLNKSQSALGTAIERLSSGLRINSAKDDAAGQAIANRFTANIKGLTQASRNANDGISIAQTTEGALNEINNNLQRVRELAVQSANSTNSQSDLDSIQAEITQRLNEIDRVSGQTQFNGVKVLAQDNTLTIQVGANDGETIDIDLKQINSQTLGLDSLNVQKAYDVKDTAVTTKAYANNGTTLDVSGLDDAAIKAATGGTNGTASVTGGAVKFDADNNKYFVTIGGFTGADAAKNGDYEVNVATDGTVTLAAGATKTTMPAGATTKTEVQELKDTPAVVSADAKNALIAGGVDATDANGAELVKMSYTDKNGKTIEGGYALKAGDKYYAADYDEATGAIKAKTTSYTAADGTTKTAANQLGGVDGKTEVVTIDGKTYNASKAAGHDFKAQPELAEAAAKTTENPLQKIDAALAQVDALRSDLGAVQNRFNSAITNLGNTVNNLSEARSRIEDSDYATEVSNMSRAQILQQAGTSVLAQANQVPQNVLSLLRLSLLTEVETPIRNEWGSRSNDSSDPLESLLTEVETPIRNEWGSRSNDSSDPGSSLLTEVETPIRNEWGSRSNDSSDPELSLLTEVETPIRNEWGSRSNDSSDPSR STF2.HA1-2(VN) SEQ ID NO: 95MAQVINTNSLSLLTQNNLNKSQSALGTAIERLSSGLRINSAKDDAAGQAIANRFTANIKGLTQASRNANDGISIAQTTEGALNEINNNLQRVRELAVQSANSTNSQSDLDSIQAEITQRLNEIDRVSGQTQFNGVKVLAQDNTLTIQVGANDGETIDIDLKQINSQTLGLDSLNVQKAYDVKDTAVTTKAYANNGTTLDVSGLDDAAIKAATGGTNGTASVTGGAVKFDADNNKYFVTIGGFTGADAAKNGDYEVNVATDGTVTLAAGATKTTMPAGATTKTEVQELKDTPAVVSADAKNALIAGGVDATDANGAELVKMSYTDKNGKTIEGGYALKAGDKYYAADYDEATGAIKAKTTSYTAADGTTKTAANQLGGVDGKTEVVTIDGKTYNASKAAGHDFKAQPELAEAAAKTTENPLQKIDAALAQVDALRSDLGAVQNRFNSAITNLGNTVNNLSEARSRIEDSDYATEVSNMSRAQILQQAGTSVLAQANQVPQNVLSLLAGVKPLILRDCSVAGWLLGNPMCDEFINVPEWSYIVEKANPVNDLCYPGDFNDYEELKHLLSRINHFEKIQIIPKSSWSSHEASLGVSSACPYQGKSSFFRNVVWLIKKNSTYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQTKLYQNPTTYISVGTSTLNQRLVPRIATRSKVNGQSGRMEFFWTILKPNDAINFESNGNFIAPEYAYKIVKKGDSTIMKSE STF2 SEQ ID NO: 96MAQVINTNSLSLLTQNNLNKSQSALGTAIERLSSGLRINSAKDDAAGQAIANRFTANIKGLTQASRNANDGISIAQTTEGALNEINNNLQRVRELAVQSANSTNSQSDLDSIQAEITQRLNEIDRVSGQTQFNGVKVLAQDNTLTIQVGANDGETIDIDLKQINSQTLGLDSLNVQKAYDVKDTAVTTKAYANNGTTLDVSGLDDAAIKAATGGTNGTASVTGGAVKFDADNNKYFVTIGGFTGADAAKNGDYEVNVATDGTVTLAAGATKTTMPAGATTKTEVQELKDTPAVVSADAKNALIAGGVDATDANGAELVKMSYTDKNGKTIEGGYALKAGDKYYAADYDEATGAIKAKTTSYTAADGTTKTAANQLGGVDGKTEVVTIDGKTYNASKAAGHDFKAQPELAEAAAKTTENPLQKIDAALAQVDALRSDLGAVQNRFNSAITNLGNTVNNLSEARSRIEDSDYATEVSNMSRAQILQQAGTSVLAQANQVPQNVLSLLA STF2.Blp.wtSEQ ID NO: 97ATGAAATTCTTAGTCAACGTTGCCCTTGTTTTTATGGTCGTATACATTTCTTACATCTATGCGGCCCAGGTTATCAATACCAACTCCCTGTCGTTGCTCACCCAAAATAACCTTAATAAAAGCCAGAGCGCACTGGGAACCGCCATAGAACGCCTCTCAAGCGGCCTCCGGATCAATTCTGCAAAAGACGACGCCGCCGGTCAGGCCATCGCAAACCGCTTTACCGCCAATATCAAGGGACTGACGCAGGCTTCGAGGAATGCTAACGATGGAATAAGCATCGCTCAAACCACGGAGGGCGCCCTGAACGAGATCAACAACAACCTACAGCGCGTCAGGGAGCTCGCAGTGCAGTCCGCCAATTCGACCAACTCGCAGTCGGACCTGGACTCGATCCAAGCCGAAATCACCCAGCGCCTGAATGAGATTGACCGGGTGAGCGGTCAGACACAGTTTAACGGCGTGAAGGTACTTGCACAGGATAACACACTTACGATACAGGTGGGCGCCAACGATGGTGAAACCATAGACATTGATCTCAAACAGATTAACAGCCAGACGCTCGGGTTGGATAGCCTGAATGTGCAAAAGGCGTACGACGTGAAAGACACGGCGGTCACTACCAAAGCCTACGCTAACAATGGCACTACCTTGGATGTGAGCGGATTGGATGATGCAGCAATCAAGGCTGCTACCGGCGGTACGAACGGAACCGCGTCCGTGACCGGCGGTGCCGTGAAGTTCGATGCTGACAACAATAAGTATTTCGTCACCATTGGAGGCTTTACTGGCGCCGACGCAGCAAAGAACGGCGACTATGAAGTGAACGTGGCAACCGATGGAACCGTGACGCTGGCCGCTGGTGCCACCAAGACCACCATGCCAGCCGGCGCCACAACTAAGACCGAGGTGCAGGAGTTAAAGGACACCCCCGCGGTGGTTAGCGCAGATGCCAAAAACGCGTTGATCGCCGGCGGAGTGGATGCAACTGATGCTAATGGTGCGGAGCTGGTTAAAATGTCGTATACAGACAAGAATGGTAAGACGATCGAGGGCGGTTATGCCCTTAAGGCAGGAGATAAGTATTACGCTGCTGATTACGATGAGGCGACGGGAGCTATTAAGGCCAAGACAACGTCATACACGGCGGCGGACGGAACGACTAAGACGGCTGCCAATCAGTTGGGAGGGGTTGACGGGAAGACAGAGGTCGTTACGATCGATGGCAAGACATACAACGCCTCCAAGGCCGCTGGCCACGATTTCAAAGCTCAACCCGAACTGGCCGAGGCCGCGGCGAAAACAACTGAGAACCCGTTGCAGAAGATTGATGCGGCCCTGGCGCAAGTAGATGCCCTGCGCTCAGACCTGGGCGCCGTTCAAAATCGATTCAATTCCGCGATTACAAACCTGGGCAATACAGTAAACAATCTATCCGAGGCCAGATCCCGCATTGAAGACTCCGACTACGCGACAGAAGTAAGTAACATGAGTCGTGCCCAGATTCTGCAGCAGGCCGGCACTAGTGTCCTGGCCCAGGCCAATCAAGTCCCGCAGAATGTGCTGAGCCTACTACGAGAGTTCAGTCGTTACCCAGCCCAATGGCGGCCGCTC STF2.Blp.ngSEQ ID NO: 98ATGAAATTCTTAGTCAACGTTGCCCTTGTTTTTATGGTCGTATACATTTCTTACATCTATGCGGCCCAAGTGATCAACACCAACTCCCTGTCCCTGCTCACTCAAAACAACCTCCAGAAATCTCAGTCTGCTCTCGGTACTGCTATCGAGCGTCTCTCCTCCGGCCTGAGGATCAACTCCGCTAAAGATGACGCCGCCGGACAAGCTATCGCTAACCGCTTTACTGCCAACATTAAAGGCCTGACCCAAGCCTCTAGGAACGCCAATGATGGCATTTCTATCGCTCAAACCACCGAAGGCGCCCTGAACGAAATCAATAATAACCTGCAACGTGTCCGCGAACTCGCCGTCCAGTCCGCCCAATCTACCAACTCTCAGTCTGACCTGGACTCTATCCAAGCTGAGATCACCCAAAGGCTCAACGAGATCGATCGTGTGTCTGGTCAAACTCAGTTTAACGGCGTCAAAGTGCTGGCTCAAGACAACACACTCACCATCCAAGTAGGAGCCAATGACGGCGAGACAATCGATATCGACCTGAAGCAGATCAATTCTCAAACTCTGGGCCTGGACTCCCTGAACGTCCAAAAGGCTTACGACGTGAAGGACACCGCTGTGACAACCAAAGCCTATGCTAATCAAGGAACAACCCTGGACGTGTCCGGACTCGATGACGCCGCTATCAAGGCCGCTACTGGCGGCACTCAGGGAACTGCCTCTGTGACCGGAGGCGCTGTGAAGTTCGACGCCGATAACAACAAATACTTCGTCACTATTGGTGGTTTCACTGGCGCTGACGCCGCTAAGAACGGTGACTACGAGGTCAACGTCGCCACTGACGGAACAGTGACACTGGCCGCTGGCGCTACCAAGACCACAATGCCTGCTGGTGCTACTACTAAGACAGAGGTGCAGGAACTCAAGGACACCCCTGCCGTGGTGTCCGCCGATGCTAAGAACGCTCTCATTGCTGGTGGTGTCGACGCTACGGACGCCAACGGAGCCGAGCTTGTGAAAATGTCCTACACCGACAAGAACGGAAAGACTATCGAGGGAGGTTACGCTCTGAAGGCTGGCGATAAGTACTACGCCGCTGATTATGACGAAGCTACAGGCGCCATTAAAGCTAAAACCACATCTTATACCGCTGCCGATGGTACTACCAAGACTGCTGCCAATCAGCTGGGTGGAGTCGATGGAAAAACCGAAGTGGTCACAATCGACGGAAAGACCTATCAAGCCTCCAAGGCTGCTGGCCACGACTTCAAGGCCCAACCCGAACTGGCCGAGGCTGCTGCTAAAACCACTGAAAACCCCCTGCAGAAAATTGACGCTGCCCTGGCCCAAGTGGATGCCCTCCGCTCCGACCTCGGCGCTGTGCAAAACCGCTTCAACTCCGCTATTACAAACCTCGGCAATACCGTCAATCAGCTCTCTGAAGCTAGGTCTCGTATTGAGGATTCCGATTACGCTACCGAGGTCTCCCAGATGTCCCGTGCCCAAATTCTCCAACAAGCCGGCACCTCCGTCCTCGCCCAAGCCAATCAGGTGCCACAGAAT GTGCTGAGCHoney Bee mellitin SEQ ID NO: 99ATGAAATTCTTAGTCAACGTTGCCCTTGTTTTTATGGTCGTATACATTTCTTACATCTAT GCGSTF2.HA0s SEQ ID NO: 100ATGAAATTCTTAGTCAACGTTGCCCTTGTTTTTATGGTCGTATACATTTCTTACATCTATGCGGCCCAGGTTATCAATACCAACTCCCTGTCGTTGCTCACCCAAAATAACCTTAATAAAAGCCAGAGCGCACTGGGAACCGCCATAGAACGCCTCTCAAGCGGCCTCCGGATCAATTCTGCAAAAGACGACGCCGCCGGTCAGGCCATCGCAAACCGCTTTACCGCCAATATCAAGGGACTGACGCAGGCTTCGAGGAATGCTAACGATGGAATAAGCATCGCTCAAACCACGGAGGGCGCCCTGAACGAGATCAACAACAACCTACAGCGCGTCAGGGAGCTCGCAGTGCAGTCCGCCAATTCGACCAACTCGCAGTCGGACCTGGACTCGATCCAAGCCGAAATCACCCAGCGCCTGAATGAGATTGACCGGGTGAGCGGTCAGACACAGTTTAACGGCGTGAAGGTACTTGCACAGGATAACACACTTACGATACAGGTGGGCGCCAACGATGGTGAAACCATAGACATTGATCTCAAACAGATTAACAGCCAGACGCTCGGGTTGGATAGCCTGAATGTGCAAAAGGCGTACGACGTGAAAGACACGGCGGTCACTACCAAAGCCTACGCTAACAATGGCACTACCTTGGATGTGAGCGGATTGGATGATGCAGCAATCAAGGCTGCTACCGGCGGTACGAACGGAACCGCGTCCGTGACCGGCGGTGCCGTGAAGTTCGATGCTGACAACAATAAGTATTTCGTCACCATTGGAGGCTTTACTGGCGCCGACGCAGCAAAGAACGGCGACTATGAAGTGAACGTGGCAACCGATGGAACCGTGACGCTGGCCGCTGGTGCCACCAAGACCACCATGCCAGCCGGCGCCACAACTAAGACCGAGGTGCAGGAGTTAAAGGACACCCCCGCGGTGGTTAGCGCAGATGCCAAAAACGCCTTGATCGCCGGCGGAGTGGATGCAACTGATGCTAATGGTGCGGAGCTGGTTAAAATGTCGTATACAGACAAGAATGGTAAGACGATCGAGGGCGGTTATGCCCTTAAGGCAGGAGATAAGTATTACGCTGCTGATTACGATGAGGCGACGGGAGCTATTAAGGCCAAGACAACGTCATACACGGCGGCGGACGGAACGACTAAGACGGCTGCCAATCAGTTGGGAGGGGTTGACGGGAAGACAGAGGTCGTTACGATCGATGGCAAGACATACAACGCTTCCAAGGCCGCTGGCCACGATTTCAAAGCTCAACCCGAACTGGCCGAGGCCGCGGCGAAAACAACTGAGAACCCGTTGCAGAAGATTGATGCGGCCCTGGCGCAAGTAGATGCCCTGCGCTCAGACCTGGGCGCCGTTCAAAATCGATTCAATTCCGCGATTACAAACCTGGGCAATACAGTAAACAATCTATCCGAGGCCAGATCCCGCATTGAAGACTCCGACTACGCGACAGAAGTAAGTAACATGAGTCGTGCCCAGATTCTGCAGCAGGCCGGCACTAGTGTCCTGGCCCAGGCCAATCAAGTCCCGCAGAATGTGCTGAGCCTACTAGCATCAGGTTCCGGCTCAGGTTCCGACACCATTTGCATTGGATACCATGCAAACAACTCAACCGATACTGTTGATACCGTCCTTGAGAAGAACGTTACCGTCACGCACTCGGTCAACCTATTAGAGGATAGCCACAACGGAAAGCTGTGCCGTCTGAAAGGCATAGCGCCACTGCAGCTCGGCAAATGTAACATCGCAGGTTGGCTCCTTGGTAACCCGGAGTGCGACCCCCTCCTCCCTGTACGATCCTGGAGTTATATCGTGGAGACCCCCAATAGCGAGAACGGAATTTGCTACCCAGGAGATTTCATAGACTACGAGGAGTTGCGCGAGCAGCTTTCGTCTGTGAGCAGCTTCGAAAGGTTCGAGATATTCCCGAAGGAGAGCAGCTGGCCGAATCATAACACTAACGGTGTGACAGCCGCCTGCAGTCATGAAGGAAAGAGTTCATTCTATCGCAACCTGCTGTGGTTGACGGAGAAAGAGGGCAGCTACCCTAAGTTGAAGAACTCCTATGTGAACAAAAAAGGCAAGGAGGTTCTGGTGCTGTGGGGCATACACCACCCCCCCAATAGCAAGGAGCAGCAGAATCTGTACCAAAACGAGAATGCCTATGTGAGCGTGGTCACTAGTAACTATAACCGTCGGTTCACTCCCGAGATCGCCGAGCGTCCGAAGGTGAGGGACCAGGCAGGCCGGATGAACTACTACTGGACCCTATTGAAGCCAGGGGACACGATTATCTTCGAGGCAAACGGAAACCTCATAGCGCCGATGTACGCCTTCGCCCTGAGCCGTGGCTTTGGATCGGGGATCATCACGTCTAACGCCTCGATGCACGAATGTAATACCAAATGCCAGACCCCACTGGGTGCTATCAACTCGTCCTTACCCTATCAAAATATACATCCGGTCACCATAGGCGAGTGTCCCAAATATGTCAGATCCGCCAAGTTGCGGATGGTGACCGGCCTCCGCAATATTCCTAGTATTCAGTCACGCGGCTTGTTCGGCGCCATCGCTGGTTTCATCGAAGGCGGGTGGACAGGCATGATTGATGGCTGGTATGGCTATCACCACCAGAACGAGCAGGGCTCGGGCTACGCGGCTGACCAGAAGTCGACTCAGAATGCCATCAATGGCATCACGAATAAGGTGAACACGGTCATTGAAAAGATGAACATTCAATTTACAGCCGTAGGAAAAGAGTTTAATAAACTGGAAAAAAGAATGGAGAATCTGAATAAGAAGGTGGACGACGGATTTTTGGACATCTGGACGTACAACGCCGAGCTGCTGGTTCTGCTGGAAAATGAGCGAACACTGGATTTTCATGATTCTAACGTAAAGAATCTGTACGAGAAGGTGAAGTCCCAACTAAAGAATAATGCCAAGGAAATCGGAAATGGATGCTTTGAGTTTTACCACAAGTGCGATAATGAGTGCATGGAATCCGTGCGAAATGGTACATACGATTACCCAAAGTACTCCGAAGAATCCAAGCTAAATCGCGAAAAGGTTGATGGTGTTAAACTTGAATCCATGGGTATTTACCAACACCATCATCACCACCATTAATAG FOR PRIMER SEQ ID NO: 101AGTCGCTGAGCCTACTAGCATCAGGTTCCGGCTCAGGTTCCGACACCATTTGCATTGGAT ACCATGCREV PRIMER SEQ ID NO: 102AGTCGCATGCCTATTAATGGTGGTGATGATGGTGTTGGTAAATACCCATGGATTC HA0s PR8SEQ ID NO: 103GACACCATTTGCATTGGATACCATGCAAACAACTCAACCGATACTGTTGATACCGTCCTTGAGAAGAACGTTACCGTCACGCACTCGGTCAACCTATTAGAGGATAGCCACAACGGAAAGCTGTGCCGTCTGAAAGGCATAGCGCCACTGCAGCTCGGCAAATGTAACATCGCAGGTTGGCTCCTTGGTAACCCGGAGTGCGACCCCCTCCTCCCTGTACGATCCTGGAGTTATATCGTGGAGACCCCCAATAGCGAGAACGGAATTTGCTACCCAGGAGATTTCATAGACTACGAGGAGTTGCGCGAGCAGCTTTCGTCTGTGAGCAGCTTCGAAAGGTTCGAGATATTCCCGAAGGAGAGCAGCTGGCCGAATCATAACACTAACGGTGTGACAGCCGCCTGCAGTCATGAAGGAAAGAGTTCATTCTATCGCAACCTGCTGTGGTTGACGGAGAAAGAGGGCAGCTACCCTAAGTTGAAGAACTCCTATGTGAACAAAAAAGGCAAGGAGGTTCTGGTGCTGTGGGGCATACACCACCCCCCCAATAGCAAGGAGCAGCAGAATCTGTACCAAAACGAGAATGCCTATGTGAGCGTGGTCACTAGTAACTATAACCGTCGGTTCACTCCCGAGATCGCCGAGCGTCCGAAGGTGAGGGACCAGGCAGGCCGGATGAACTACTACTGGACCCTATTGAAGCCAGGGGACACGATTATCTTCGAGGCAAACGGAAACCTCATAGCGCCGATGTACGCCTTCGCCCTGAGCCGTGGCTTTGGATCGGGGATCATCACGTCTAACGCCTCGATGCACGAATGTAATACCAAATGCCAGACCCCACTGGGTGCTATCAACTCGTCCTTACCCTATCAAAATATACATCCGGTCACCATAGGCGAGTGTCCCAAATATGTCAGATCCGCCAAGTTGCGGATGGTGACCGGCCTCCGCAATATTCCTAGTATTCAGTCACGCGGCTTGTTCGGCGCCATCGCTGGTTTCATCGAAGGCGGGTGGACAGGCATGATTGATGGCTGGTATGGCTATCACCACCAGAACGAGCAGGGCTCGGGCTACGCGGCTGACCAGAAGTCGACTCAGAATGCCATCAATGGCATCACGAATAAGGTGAACACGGTCATTGAAAAGATGAACATTCAATTTACAGCCGTAGGAAAAGAGTTTAATAAACTGGAAAAAAGAATGGAGAATCTGAATAAGAAGGTGGACGACGGATTTTTGGACATCTGGACGTACAACGCCGAGCTGCTGGTTCTGCTGGAAAATGAGCGAACACTGGATTTTCATGATTCTAACGTAAAGAATCTGTACGAGAAGGTGAAGTCCCAACTAAAGAATAATGCCAAGGAAATCGGAAATGGATGCTTTGAGTTTTACCACAAGTGCGATAATGAGTGCATGGAATCCGTGCGAAATGGTACATACGATTACCCAAAGTACTCCGAAGAATCCAAGCTAAATCGCGAAAAGGTTGATGGTGTTAAACTTGAATCCATGGGTATTTACCAACACCATCATCACCACCATTAATAG STF2.HA1-1SEQ ID NO: 104ATGAAATTCTTAGTCAACGTTGCCCTTGTTTTTATGGTCGTATACATTTCTTACATCTATGCGGCCCAGGTTATCAATACCAACTCCCTGTCGTTGCTCACCCAAAATAACCTTAATAAAAGCCAGAGCGCACTGGGAACCGCCATAGAACGCCTCTCAAGCGGCCTCCGGATCAATTCTGCAAAAGACGACGCCGCCGGTCAGGCCATCGCAAACCGCTTTACCGCCAATATCAAGGGACTGACGCAGGCTTCGAGGAATGCTAACGATGGAATAAGCATCGCTCAAACCACGGAGGGCGCCCTGAACGAGATCAACAACAACCTACAGCGCGTCAGGGAGCTCGCAGTGCAGTCCGCCAATTCGACCAACTCGCAGTCGGACCTGGACTCGATCCAAGCCGAAATCACCCAGCGCCTGAATGAGATTGACCGGGTGAGCGGTCAGACACAGTTTAACGGCGTGAAGGTACTTGCACAGGATAACACACTTACGATACAGGTGGGCGCCAACGATGGTGAAACCATAGACATTGATCTCAAACAGATTAACAGCCAGACGCTCGGGTTGGATAGCCTGAATGTGCAAAAGGCGTACGACGTGAAAGACACGGCGGTCACTACCAAAGCCTACGCTAACAATGGCACTACCTTGGATGTGAGCGGATTGGATGATGCAGCAATCAAGGCTGCTACCGGCGGTACGAACGGAACCGCGTCCGTGACCGGCGGTGCCGTGAAGTTCGATGCTGACAACAATAAGTATTTCGTCACCATTGGAGGCTTTACTGGCGCCGACGCAGCAAAGAACGGCGACTATGAAGTGAACGTGGCAACCGATGGAACCGTGACGCTGGCCGCTGGTGCCACCAAGACCACCATGCCAGCCGGCGCCACAACTAAGACCGAGGTGCAGGAGTTAAAGGACACCCCCGCGGTGGTTAGCGCAGATGCCAAAAACGCCTTGATCGCCGGCGGAGTGGATGCAACTGATGCTAATGGTGCGGAGCTGGTTAAAATGTCGTATACAGACAAGAATGGTAAGACGATCGAGGGCGGTTATGCCCTTAAGGCAGGAGATAAGTATTACGCTGCTGATTACGATGAGGCGACGGGAGCTATTAAGGCCAAGACAACGTCATACACGGCGGCGGACGGAACGACTAAGACGGCTGCCAATCAGTTGGGAGGGGTTGACGGGAAGACAGAGGTCGTTACGATCGATGGCAAGACATACAACGCTTCCAAGGCCGCTGGCCACGATTTCAAAGCTCAACCCGAACTGGCCGAGGCCGCGGCGAAAACAACTGAGAACCCGTTGCAGAAGATTGATGCGGCCCTGGCGCAAGTAGATGCCCTGCGCTCAGACCTGGGCGCCGTTCAAAATCGATTCAATTCCGCGATTACAAACCTGGGCAATACAGTAAACAATCTATCCGAGGCCAGATCCCGCATTGAAGACTCCGACTACGCGACAGAAGTAAGTAACATGAGTCGTGCCCAGATTCTGCAGCAGGCCGGCACTAGTGTCCTGGCCCAGGCCAATCAAGTCCCGCAGAATGTGCTGAGCCTACTAGCATCAGGTTCCGGCTCAGGTTCCAGCCACAACGGAAAGCTGTGCCGTCTGAAAGGCATAGCGCCACTGCAGCTCGGCAAATGTAACATCGCAGGTTGGCTCCTTGGTAACCCGGAGTGCGACCCCCTCCTCCCTGTACGATCCTGGAGTTATATCGTGGAGACCCCCAATAGCGAGAACGGAATTTGCTACCCAGGAGATTTCATAGACTACGAGGAGTTGCGCGAGCAGCTTTCGTCTGTGAGCAGCTTCGAAAGGTTCGAGATATTCCCGAAGGAGAGCAGCTGGCCGAATCATAACACTAACGGTGTGACAGCCGCCTGCAGTCATGAAGGAAAGAGTTCATTCTATCGCAACCTGCTGTGGTTGACGGAGAAAGAGGGCAGCTACCCTAAGTTGAAGAACTCCTATGTGAACAAAAAAGGCAAGGAGGTTCTGGTGCTGTGGGGCATACACCACCCCCCCAATAGCAAGGAGCAGCAGAATCTGTACCAAAACGAGAATGCCTATGTGAGCGTGGTCACTAGTAACTATAACCGTCGGTTCACTCCCGAGATCGCCGAGCGTCCGAAGGTGAGGGACCAGGCAGGCCGGATGAACTACTACTGGACCCTATTGAAGCCAGGGGACACGATTATCTTCGAGGCAAACGGAAACCTCATAGCGCCGATGTACGCCTTCGCCCTGAGCCGTGGCTTTGGATCGGGGATCATCACGTCTAACGCCTCGATGCACGAATGTAATACCAAATGCCAGACCCCACTGGGTGCTATCAACTCGTCCTTACCCTATCAAAATATACATCCGGTCACCATAGGCGAGTGTCCCAAATATGTCAGACACCATCATCACCACCATTAATAG FOR PRIMER SEQ ID NO: 105AGTCGCTGAGCCTACTAGCATCAGGTTCCGGCTCAGGTTCCAGCCACAACGGAAAGCTGT GCCGTCREV PRIMER SEQ ID NO: 106AGTCGCATGCCTATTAATGGTGGTGATGATGGTGTCTGACATATTTGGGACACTCGCCTA TGGTGACSTF2.HA1-2 SEQ ID NO: 107ATGAAATTCTTAGTCAACGTTGCCCTTGTTTTTATGGTCGTATACATTTCTTACATCTATGCGGCCCAGGTTATCAATACCAACTCCCTGTCGTTGCTCACCCAAAATAACCTTAATAAAAGCCAGAGCGCACTGGGAACCGCCATAGAACGCCTCTCAAGCGGCCTCCGGATCAATTCTGCAAAAGACGACGCCGCCGGTCAGGCCATCGCAAACCGCTTTACCGCCAATATCAAGGGACTGACGCAGGCTTCGAGGAATGCTAACGATGGGATAAGCATCGCTCAAACCACGGAGGGCGCCCTGAACGAGATCAACAACAACCTACAGCGCGTCAGGGAGCTCGCAGTGCAGTCCGCCAATTCGACCAACTCGCAGTCGGACCTGGACTCGATCCAAGCCGAAATCACCCAGCGCCTGAACGAGATTGACCGGGTGAGCGGTCAGACACAGTTTAACGGCGTGAAGGTACTTGCGCAGGATAACACACTTACGATACAGGTGGGCGCCAACGATGGTGAAACCATAGACATTGATCTCAAACAGATTAACAGCCAGACGCTCGGGTTGGATAGCCTGAATGTGCAAAAGGCGTACGACGTGAAAGACACGGCGGTCACTACCAAAGCCTACGCTAACAATGGCACTACCTTGGATGTGAGCGGATTGGATGATGCAGCAATCAAGGCTGCTACCGGCGGTACGAACGGAACCGCGTCCGTGACCGGCGGTGCCGTGAAGTTCGATGCTGACAACAATAAGTATTTCGTCACCATTGGAGGCTTTACTGGCGCCGACGCAGCAAAGAACGGCGACTATGAAGTGAACGTGGCAACCGATGGAACCGTGACGCTGGCCGCTGGTGCCACCAAGACCACCATGCCAGCCGGCGCCACAACTAAGACCGAGGTGCAGGAGTTAAAGGACACCCCCGCGGTGGTTAGCGCAGATGCCAAAAACGCGTTGATCGCCGGCGGAGTGGATGCAACTGATGCTAATGGTGCGGAGCTGGTTAAAATGTCGTATACAGACAAGAATGGTAAGACGATCGAGGGCGGTTATGCCCTTAAGGCAGGAGATAAGTATTACGCTGCTGATTACGATGAGGCGACGGGAGCTATTAAGGCCAAGACAACGTCATACACGGCGGCGGACGGAACGACTAAGACGGCTGCCAATCAGTTGGGAGGGGTTGACGGGAAGACAGAGGTCGTTACGATCGATGGCAAGACATACAACGCCTCCAAGGCCGCTGGCCACGATTTCAAAGCTCAACCCGAACTGGCCGAGGCCGCGGCGAAAACAACTGAGAACCCGTTGCAGAAGATTGATGCGGCCCTGGCGCAAGTAGATGCCCTGCGCTCAGACCTGGGCGCCGTTCAAAATCGATTCAATTCCGCGATTACAAACCTGGGCAATACAGTAAACAATCTATCCGAGGCCAGATCCCGCATTGAAGACTCCGACTACGCGACAGAAGTAAGTAACATGAGTCGTGCCCAGATTCTGCAGCAGGCCGGCACTAGTGTCCTGGCCCAGGCCAATCAAGTCCCGCAGAATGTGCTGAGCCTACTAGCATCAGGTTCCGGCTCAGGTTCCAAAGGCATAGCGCCACTGCAGCTCGGCAAATGTAACATCGCAGGTTGGCTCCTTGGTAACCCGGAGTGCGACCCCCTCCTCCCTGTACGATCCTGGAGTTATATCGTGGAGACCCCCAATAGCGAGAACGGAATTTGCTACCCAGGAGATTTCATAGACTACGAGGAGTTGCGCGAGCAGCTTTCGTCTGTGAGCAGCTTCGAGAGGTTCGAAATCTTCCCGAAGGAGAGCAGCTGGCCGAATCATAACACTAACGGTGTGACAGCCGCCTGCAGTCATGAAGGAAAGAGTTCATTCTATCGCAACCTGCTGTGGTTGACGGAGAAAGAGGGCAGCTACCCTAAGTTGAAGAACTCCTATGTGAACAAAAAAGGCAAGGAGGTTCTGGTGCTGTGGGGCATACACCACCCCCCCAATAGCAAGGAGCAGCAGAATCTGTACCAAAACGAGAATGCCTATGTGAGCGTGGTCACTAGTAACTATAACCGTCGGTTCACTCCCGAGATCGCCGAGCGTCCGAAGGTGAGGGACCAGGCAGGCCGGATGAACTACTACTGGACCCTATTGAAGCCAGGGGACACGATTATCTTCGAGGCAAACGGAAACCTCATAGCGCCGATGTACGCGTTCGCCCTGAGCCGTGGCTTTGGATCGGGGATCATCACGTCTCACCATCATCACCACCATTAATAG FOR PRIMER SEQ ID NO: 108AGTCGCTGAGCCTACTAGCATCAGGTTCCGGCTCAGGTTCCAAAGGCATAGCGCCACTGC AGCTCGREV PRIMER SEQ ID NO: 109AGTCGCATGCCTATTAATGGTGGTGATGATGGTGAGACGTGATGATCCCCGATCCAAAGC CACGGCTCAGSTF2.HA1-2mut SEQ ID NO: 110ATGAAATTCTTAGTCAACGTTGCCCTTGTTTTTATGGTCGTATACATTTCTTACATCTATGCGGCCCAGGTTATCAATACCAACTCCCTGTCGTTGCTCACCCAAAATAACCTTAATAAAAGCCAGAGCGCACTGGGAACCGCCATAGAACGCCTCTCAAGCGGCCTCCGGATCAATTCTGCAAAAGACGACGCCGCCGGTCAGGCCATCGCAAACCGCTTTACCGCCAATATCAAGGGACTGACGCAGGCTTCGAGGAATGCTAACGATGGAATAAGCATCGCTCAAACCACGGAGGGCGCCCTGAACGAGATCAACAACAACCTACAGCGCGTCAGGGAGCTCGCAGTGCAGTCCGCCAATTCGACCAACTCGCAGTCGGACCTGGACTCGATCCAAGCCGAAATCACCCAGCGCCTGAATGAGATTGACCGGGTGAGCGGTCAGACACAGTTTAACGGCGTGAAGGTACTTGCACAGGATAACACACTTACGATACAGGTGGGCGCCAACGATGGTGAAACCATAGACATTGATCTCAAACAGATTAACAGCCAGACGCTCGGGTTGGATAGCCTGAATGTGCAAAAGGCGTACGACGTGAAAGACACGGCGGTCACTACCAAAGCCTACGCTAACAATGGCACTACCTTGGATGTGAGCGGATTGGATGATGCAGCAATCAAGGCTGCTACCGGCGGTACGAACGGAACCGCGTCCGTGACCGGCGGTGCCGTGAAGTTCGATGCTGACAACAATAAGTATTTCGTCACCATTGGAGGCTTTACTGGCGCCGACGCAGCAAAGAACGGCGACTATGAAGTGAACGTGGCAACCGATGGAACCGTGACGCTGGCCGCTGGTGCCACCAAGACCACCATGCCAGCCGGCGCCACAACTAAGACCGAGGTGCAGGAGTTAAAGGACACCCCCGCGGTGGTTAGCGCAGATGCCAAAAACGCGTTGATCGCCGGCGGAGTGGATGCAACTGATGCTAATGGTGCGGAGCTGGTTAAAATGTCGTATACAGACAAGAATGGTAAGACGATCGAGGGCGGTTATGCCCTTAAGGCAGGAGATAAGTATTACGCTGCTGATTACGATGAGGCGACGGGAGCTATTAAGGCCAAGACAACGTCATACACGGCGGCGGACGGAACGACTAAGACGGCTGCCAATCAGTTGGGAGGGGTTGACGGGAAGACAGAGGTCGTTACGATCGATGGCAAGACATACAACGCCTCCAAGGCCGCTGGCCACGATTTCAAAGCTCAACCCGAACTGGCCGAGGCCGCGGCGAAAACAACTGAGAACCCGTTGCAGAAGATTGATGCGGCCCTGGCGCAAGTAGATGCCCTGCGCTCAGACCTGGGCGCCGTTCAAAATCGATTCAATTCCGCGATTACAAACCTGGGCAATACAGTAAACAATCTATCCGAGGCCAGATCCCGCATTGAAGACTCCGACTACGCGACAGAAGTAAGTAACATGAGTCGTGCCCAGATTCTGCAGCAGGCCGGCACTAGTGTCCTGGCCCAGGCCAATCAAGTCCCGCAGAATGTGCTGAGCCTGCTGGCTTCTGGATCTGGATCTGGTTCTAAAGGCGCTGCCCCTCTGCAACTCGGCAAGTGCAATATTGCGGGGTGGCTGTTGGGCAACCCAGAATGTGACCCCCTCCTGCCCGTCCGTTCTTGGTCTGACATCGCTGAAACACCTAACTCCGAGAACGGCATCTGTTACCCGGGCGACTTCATTGACTACGAGGAGCTCCGCGAACAGCTGTCTTCTGTCTCTTCTTTCGAACGTTTCGAAATTTTCCCAAAGGAATCCTCCTGGCCAAACCATAACACCAACGGAGTGACCGCCGCCTGTAGCCATGAGGGCAAGTCTTCTTTCTACCGTAATCTGCTGTGGCTGACTGAAAAAGAGGGTTCTTATCCCAAACTGAAGAACTCTTATGTCAACAAGAAGGGCAAAGAGGTCCTGGTGCTGTGGGGTATCCACCACCCCCCCAACTCCAAGGAGCAGCAGAATCTGTACCAAAACGAAAATGCTTACGTGTCTGTCGTGACATCTAACTACAACCGGCGCTTCACGCCCGAAATCGCCGAGCGTCCCAAAGTGCGCGATCAGGCCGGAAGGATGAACTACTACTGGACCCTGCTCAAACCCGGAGATACCATCATATTCGAAGCCAATGGCAATCTCATCGCTCCCATGTATGCTTTCGCTCTCTCTCGCGGATTCGGTTCCGGCATAATCACTAGCCACCACCACCACCACCACTAATAG HA1-2mut SEQ ID NO: 111AAGGCGCTGCCCCTCTGCAACTCGGCAAGTGCAATATTGCGGGGTGGCTGTTGGGCAACCCAGAATGTGACCCCCTCCTGCCCGTCCGTTCTTGGTCTGACATCGCTGAAACACCTAACTCCGAGAACGGCATCTGTTACCCGGGCGACTTCATTGACTACGAGGAGCTCCGCGAACAGCTGTCTTCTGTCTCTTCTTTCGAACGTTTCGAAATTTTCCCAAAGGAATCCTCCTGGCCAAACCATAACACCAACGGAGTGACCGCCGCCTGTAGCCATGAGGGCAAGTCTTCTTTCTACCGTAATCTGCTGTGGCTGACTGAAAAAGAGGGTTCTTATCCCAAACTGAAGAACTCTTATGTCAACAAGAAGGGCAAAGAGGTCCTGGTGCTGTGGGGTATCCACCACCCCCCCAACTCCAAGGAGCAGCAGAATCTGTACCAAAACGAAAATGCTTACGTGTCTGTCGTGACATCTAACTACAACCGGCGCTTCACGCCCGAAATCGCCGAGCGTCCCAAAGTGCGCGATCAGGCCGGAAGGATGAACTACTACTGGACCCTGCTCAAACCCGGAGATACCATCATATTCGAAGCCAATGGCAATCTCATCGCTCCCATGTATGCTTTCGCTCTCTCTCGCGGATTCGGTTCCGGCATAATCACTAGCCACCACCACCACCACCACTAATAG STF2.HA1-3 SEQ ID NO: 112ATGAAATTCTTAGTCAACGTTGCCCTTGTTTTTATGGTCGTATACATTTCTTACATCTATGCGGCCCAGGTTATCAATACCAACTCCCTGTCGTTGCTCACCCAAAATAACCTTAATAAAAGCCAGAGCGCACTGGGAACCGCCATAGAACGCCTCTCAAGCGGCCTCCGGATCAATTCTGCAAAAGACGACGCCGCCGGTCAGGCCATCGCAAACCGCTTTACCGCCAATATCAAGGGACTGACGCAGGCTTCGAGGAATGCTAACGATGGGATAAGCATCGCTCAAACCACGGAGGGCGCCCTGAACGAGATCAACAACAACCTACAGCGCGTCAGGGAGCTCGCAGTGCAGTCCGCCAATTCGACCAACTCGCAGTCGGACCTGGACTCGATCCAAGCCGAAATCACCCAGCGCCTGAACGAGATTGACCGGGTGAGCGGTCAGACACAGTTTAACGGCGTGAAGGTACTTGCGCAGGATAACACACTTACGATACAGGTGGGCGCCAACGATGGTGAAACCATAGACATTGATCTCAAACAGATTAACAGCCAGACGCTCGGGTTGGATAGCCTGAATGTGCAAAAGGCGTACGACGTGAAAGACACGGCGGTCACTACCAAAGCCTACGCTAACAATGGCACTACCTTGGATGTGAGCGGATTGGATGATGCAGCAATCAAGGCTGCTACCGGCGGTACGAACGGAACCGCGTCCGTGACCGGCGGTGCCGTGAAGTTCGATGCTGACAACAATAAGTATTTCGTCACCATTGGAGGCTTTACTGGCGCCGACGCAGCAAAGAACGGCGACTATGAAGTGAACGTGGCAACCGATGGAACCGTGACGCTGGCCGCTGGTGCCACCAAGACCACCATGCCAGCCGGCGCCACAACTAAGACCGAGGTGCAGGAGTTAAAGGACACCCCCGCGGTGGTTAGCGCAGATGCCAAAAACGCGTTGATCGCCGGCGGAGTGGATGCAACTGATGCTAATGGTGCGGAGCTGGTTAAAATGTCGTATACAGACAAGAATGGTAAGACGATCGAGGGCGGTTATGCCCTTAAGGCAGGAGATAAGTATTACGCTGCTGATTACGATGAGGCGACGGGAGCTATTAAGGCCAAGACAACGTCATACACGGCGGCGGACGGAACGACTAAGACGGCTGCCAATCAGTTGGGAGGGGTTGACGGGAAGACAGAGGTCGTTACGATCGATGGCAAGACATACAACGCCTCCAAGGCCGCTGGCCACGATTTCAAAGCTCAACCCGAACTGGCCGAGGCCGCGGCGAAAACAACTGAGAACCCGTTGCAGAAGATTGATGCGGCCCTGGCGCAAGTAGATGCCCTGCGCTCAGACCTGGGCGCCGTTCAAAATCGATTCAATTCCGCGATTACAAACCTGGGCAATACAGTAAACAATCTATCCGAGGCCAGATCCCGCATTGAAGACTCCGACTACGCGACAGAAGTAAGTAACATGAGTCGTGCCCAGATTCTGCAGCAGGCCGGCACTAGTGTCCTGGCCCAGGCCAATCAAGTCCCGCAGAATGTGCTGAGCCTACTAGCATCAGGTTCCGGCTCAGGTTCCAATAGCGAGAACGGAATTTGCTACCCAGGAGATTTCATAGACTACGAGGAGTTGCGCGAGCAGCTTTCGTCTGTGAGCAGCTTCGAGAGGTTCGAAATCTTCCCGAAGGAGAGCAGCTGGCCGAATCATAACACTAACGGTGTGACAGCCGCCTGCAGTCATGAAGGAAAGAGTTCATTCTATCGCAACCTGCTGTGGTTGACGGAGAAGGAGGGCAGCTACCCTAAGCTGAAGAACTCCTATGTGAACAAAAAAGGCAAGGAGGTTCTGGTGCTGTGGGGCATACACCACCCCCCCAATAGCAAGGAGCAGCAGAATCTGTACCAAAACGAGAATGCCTATGTGAGCGTGGTCACTAGTAACTATAACCGTCGGTTCACTCCCGAGATCGCCGAGCGTCCGAAGGTGAGGGACCAGGCAGGCCGGATGAACTACTACTGGACCCTATTGAAGCCAGGGGACACGATTATCTTCGAGGCAAACGGAAACCTCATAGCGCCGATGTACGCCTTCGCCCTGAGCCGTGGCCACCATCATCACCACCATTAATAG FOR PRIMERSEQ ID NO: 113AGTCGCTGAGCCTACTAGCATCAGGTTCCGGCTCAGGTTCCAATAGCGAGAACGGAATTT GCTACCREV PRIMER SEQ ID NO: 114AGTCGCATGCCTATTAATGGTGGTGATGATGGTGGCCACGGCTCAGGGCGAAGG STF2.HA1-3mutSEQ ID NO: 115ATGAAATTCTTAGTCAACGTTGCCCTTGTTTTTATGGTCGTATACATTTCTTACATCTATGCGGCCCAGGTTATCAATACCAACTCCCTGTCGTTGCTCACCCAAAATAACCTTAATAAAAGCCAGAGCGCACTGGGAACCGCCATAGAACGCCTCTCAAGCGGCCTCCGGATCAATTCTGCAAAAGACGACGCCGCCGGTCAGGCCATCGCAAACCGCTTTACCGCCAATATCAAGGGACTGACGCAGGCTTCGAGGAATGCTAACGATGGAATAAGCATCGCTCAAACCACGGAGGGCGCCCTGAACGAGATCAACAACAACCTACAGCGCGTCAGGGAGCTCGCAGTGCAGTCCGCCAATTCGACCAACTCGCAGTCGGACCTGGACTCGATCCAAGCCGAAATCACCCAGCGCCTGAATGAGATTGACCGGGTGAGCGGTCAGACACAGTTTAACGGCGTGAAGGTACTTGCACAGGATAACACACTTACGATACAGGTGGGCGCCAACGATGGTGAAACCATAGACATTGATCTCAAACAGATTAACAGCCAGACGCTCGGGTTGGATAGCCTGAATGTGCAAAAGGCGTACGACGTGAAAGACACGGCGGTCACTACCAAAGCCTACGCTAACAATGGCACTACCTTGGATGTGAGCGGATTGGATGATGCAGCAATCAAGGCTGCTACCGGCGGTACGAACGGAACCGCGTCCGTGACCGGCGGTGCCGTGAAGTTCGATGCTGACAACAATAAGTATTTCGTCACCATTGGAGGCTTTACTGGCGCCGACGCAGCAAAGAACGGCGACTATGAAGTGAACGTGGCAACCGATGGAACCGTGACGCTGGCCGCTGGTGCCACCAAGACCACCATGCCAGCCGGCGCCACAACTAAGACCGAGGTGCAGGAGTTAAAGGACACCCCCGCGGTGGTTAGCGCAGATGCCAAAAACGCGTTGATCGCCGGCGGAGTGGATGCAACTGATGCTAATGGTGCGGAGCTGGTTAAAATGTCGTATACAGACAAGAATGGTAAGACGATCGAGGGCGGTTATGCCCTTAAGGCAGGAGATAAGTATTACGCTGCTGATTACGATGAGGCGACGGGAGCTATTAAGGCCAAGACAACGTCATACACGGCGGCGGACGGAACGACTAAGACGGCTGCCAATCAGTTGGGAGGGGTTGACGGGAAGACAGAGGTCGTTACGATCGATGGCAAGACATACAACGCCTCCAAGGCCGCTGGCCACGATTTCAAAGCTCAACCCGAACTGGCCGAGGCCGCGGCGAAAACAACTGAGAACCCGTTGCAGAAGATTGATGCGGCCCTGGCGCAAGTAGATGCCCTGCGCTCAGACCTGGGCGCCGTTCAAAATCGATTCAATTCCGCGATTACAAACCTGGGCAATACAGTAAACAATCTATCCGAGGCCAGATCCCGCATTGAAGACTCCGACTACGCGACAGAAGTAAGTAACATGAGTCGTGCCCAGATTCTGCAGCAGGCCGGCACTAGTGTCCTGGCCCAGGCCAATCAAGTCCCGCAGAATGTGCTGAGCCTGCTGGCTTCTGGATCTGGATCTGGTTCTAACTCCGAGAACGAAATCTGTTACCCGGGCGACTTCATTGACAAAGAGGAGCTCCGCGAACAGCTGTCTTCTGTCTCTTCTTTCGAACGTTTCGAAATTTTCCCAAAGGAATCCTCCTGGCCAAACCATAACACCAACGGAGTGACCGCCGCCTGTAGCCATGAGGGCAAGTCTTCTTTCTACCGTAATCTGCTGTGGCTGACTGAAAAAGAGGGTTCTTATCCCAAACTGAAGAACTCTTATGTCAACAAGAAGGGCAAAGAGGTCCTGGTGCTGTGGGGTATCCACCACCCCCCCAACTCCAAGGAGCAGCAGAATCTGTACCAAAACGAAAATGCTTACGTGTCTGTCGTGACATCTAACTACAACCGGCGCTTCACGCCCGAAATCGCCGAGCGTCCCAAAGTGCGCGATCAGGCCGGAAGGATGAACTACTACTGGACCCTGCTCAAACCCGGAGATACCATCATATTCGAAGCCAATGGCAATCTCATCGCTCCCATGTATGCTGCTGCTCTCTCTCGCGGACACCACCACCACCACCACTAATAG HA1-3mut PR8SEQ ID NO: 116AACTCCGAGAACGAAATCTGTTACCCGGGCGACTTCATTGACAAAGAGGAGCTCCGCGAACAGCTGTCTTCTGTCTCTTCTTTCGAACGTTTCGAAATTTTCCCAAAGGAATCCTCCTGGCCAAACCATAACACCAACGGAGTGACCGCCGCCTGTAGCCATGAGGGCAAGTCTTCTTTCTACCGTAATCTGCTGTGGCTGACTGAAAAAGAGGGTTCTTATCCCAAACTGAAGAACTCTTATGTCAACAAGAAGGGCAAAGAGGTCCTGGTGCTGTGGGGTATCCACCACCCCCCCAACTCCAAGGAGCAGCAGAATCTGTACCAAAACGAAAATGCTTACGTGTCTGTCGTGACATCTAACTACAACCGGCGCTTCACGCCCGAAATCGCCGAGCGTCCCAAAGTGCGCGATCAGGCCGGAAGGATGAACTACTACTGGACCCTGCTCAAACCCGGAGATACCATCATATTCGAAGCCAATGGCAATCTCATCGCTCCCATGTATGCTGCTGCTCTCTCTCGCGGACACCACCACCACCACCACTAATAG ngSTF2.HA0s SEQ ID NO: 117ATGAAATTCTTAGTCAACGTTGCCCTTGTTTTTATGGTCGTATACATTTCTTACATCTATGCGGCCCAAGTGATCAACACCAACTCCCTGTCCCTGCTCACTCAAAACAACCTCCAGAAATCTCAGTCTGCTCTCGGTACTGCTATCGAGCGTCTCTCCTCCGGCCTGAGGATCAACTCCGCTAAAGATGACGCCGCCGGACAAGCTATCGCTAACCGCTTTACTGCCAACATTAAAGGCCTGACCCAAGCCTCTAGGAACGCCAATGATGGCATTTCTATCGCTCAAACCACCGAAGGCGCCCTGAACGAAATCAATAATAACCTGCAACGTGTCCGCGAACTCGCCGTCCAGTCCGCCCAATCTACCAACTCTCAGTCTGACCTGGACTCTATCCAAGCTGAGATCACCCAAAGGCTCAACGAGATCGATCGTGTGTCTGGTCAAACTCAGTTTAACGGCGTCAAAGTGCTGGCTCAAGACAACACACTCACCATCCAAGTAGGAGCCAATGACGGCGAGACAATCGATATCGACCTGAAGCAGATCAATTCTCAAACTCTGGGCCTGGACTCCCTGAACGTCCAAAAGGCTTACGACGTGAAGGACACCGCTGTGACAACCAAAGCCTATGCTAATCAAGGAACAACCCTGGACGTGTCCGGACTCGATGACGCCGCTATCAAGGCCGCTACTGGCGGCACTCAGGGAACTGCCTCTGTGACCGGAGGCGCTGTGAAGTTCGACGCCGATAACAACAAATACTTCGTCACTATTGGTGGTTTCACTGGCGCTGACGCCGCTAAGAACGGTGACTACGAGGTCAACGTCGCCACTGACGGAACAGTGACACTGGCCGCTGGCGCTACCAAGACCACAATGCCTGCTGGTGCTACTACTAAGACAGAGGTGCAGGAACTCAAGGACACCCCTGCCGTGGTGTCCGCCGATGCTAAGAACGCTCTCATTGCTGGTGGTGTCGACGCTACGGACGCCAACGGAGCCGAGCTTGTGAAAATGTCCTACACCGACAAGAACGGAAAGACTATCGAGGGAGGTTACGCTCTGAAGGCTGGCGATAAGTACTACGCCGCTGATTATGACGAAGCTACAGGCGCCATTAAAGCTAAAACCACATCTTATACCGCTGCCGATGGTACTACCAAGACTGCTGCCAATCAGCTGGGTGGAGTCGATGGAAAAACCGAAGTGGTCACAATCGACGGAAAGACCTATCAAGCCTCCAAGGCTGCTGGCCACGACTTCAAGGCCCAACCCGAACTGGCCGAGGCTGCTGCTAAAACCACTGAAAACCCCCTGCAGAAAATTGACGCTGCCCTGGCCCAAGTGGATGCCCTCCGCTCCGACCTCGGCGCTGTGCAAAACCGCTTCAACTCCGCTATTACAAACCTCGGCAATACCGTCAATCAACTCTCTGAAGCTAGGTCTCGTATTGAGGATTCCGATTACGCTACCGAGGTCTCCCAGATGTCCCGTGCCCAAATTCTCCAACAAGCCGGCACCTCCGTCCTCGCCCAAGCCAATCAGGTGCCACAGAATGTGCTGAGCCTGCTGGCTTCTGGATCTGGATCTGGTTCTGACACCATCTGCATTGGCTACCATGCCCAGCAGTCTACAGATACAGTCGATACCGTCCTGGAGAAACAAGTCACCGTCACGCACTCCGTGAACCTCCTGGAGGACTCCCATAATGGCAAATTGTGTCGGCTGAAAGGCATCGCCCCTCTGCAACTCGGCAAGTGCAATATTGCGGGGTGGCTGTTGGGCAACCCAGAATGTGACCCCCTCCTGCCCGTCCGTTCTTGGTCTTACATCGTGGAAACACCTAACTCCGAGAACGGCATCTGTTACCCGGGCGACTTCATTGACTACGAGGAGCTCCGCGAACAGCTGTCTTCTGTCTCTTCTTTCGAACGTTTCGAAATTTTCCCAAAGGAATCCTCCTGGCCAAACCATAACACCAACGGAGTGACCGCCGCCTGTAGCCATGAGGGCAAGTCTTCTTTCTACCGTAATCTGCTGTGGCTGACTGAAAAAGAGGGTTCTTATCCCAAACTGAAGAACTCTTATGTCAACAAGAAGGGCAAAGAGGTCCTGGTGCTGTGGGGTATCCACCACCCCCCCAACTCCAAGGAGCAGCAGAATCTGTACCAAAACGAAAATGCTTACGTGTCTGTCGTGACATCTAACTACAACCGGCGCTTCACGCCCGAAATCGCCGAGCGTCCCAAAGTGCGCGATCAGGCCGGAAGGATGAACTACTACTGGACCCTGCTCAAACCCGGAGATACCATCATATTCGAAGCCAATGGCAATCTCATCGCTCCCATGTATGCTTTCGCTCTCTCTCGCGGATTCGGTTCCGGCATAATCACTAGCCAAGCCTCCATGCACGAGTGCAATACCAAGTGTCAAACTCCACTGGGAGCCATTCAAAGCTCCCTGCCCTACCAAAACATCCATCCTGTGACCATTGGAGAGTGCCCTAAATACGTGCGCAGCGCGAAACTGCGCATGGTGACCGGTCTCCGCAATATACCCTCCATACAATCGCGCGGCCTGTTTGGTGCTATCGCTGGCTTTATCGAGGGAGGATGGACTGGAATGATCGACGGCTGGTATGGCTATCATCATCAAAACGAACAGGGTTCCGGCTACGCCGCTGACCAAAAGTCCACTCAAAACGCCATTAACGGTATTACAAACAAAGTAAACACCGTGATAGAGAAAATGAATATCCAATTCACTGCCGTGGGCAAAGAGTTTAACAAGCTGGAGAAGCGCATGGAAAATCTGAACAAAAAAGTCGATGATGGCTTCCTCGACATCTGGACTTACAACGCCGAACTCCTCGTGCTGCTCGAAAACGAGAGGACTCTGGACTTCCACGACTCCAACGTGAAGAACCTGTACGAGAAAGTCAAATCCCAACTCAAGAACAACGCCAAAGAAATCGGCAACGGCTGCTTCGAGTTCTACCACAAATGTGATAACGAGTGTATGGAATCCGTACGGCAAGGCACTTACGACTACCCCAAATACAGCGAAGAGAGCAAATTGAACCGCGAGAAAGTGGACGGCGTGAAACTGGAGTCCATGGGCATCTATCAGCACCACCACCACCACCACTAATAG ngHA0s SEQ ID NO: 118GACACCATCTGCATTGGCTACCATGCCCAGCAGTCTACAGATACAGTCGATACCGTCCTGGAGAAACAAGTCACCGTCACGCACTCCGTGAACCTCCTGGAGGACTCCCATAATGGCAAATTGTGTCGGCTGAAAGGCATCGCCCCTCTGCAACTCGGCAAGTGCAATATTGCGGGGTGGCTGTTGGGCAACCCAGAATGTGACCCCCTCCTGCCCGTCCGTTCTTGGTCTTACATCGTGGAAACACCTAACTCCGAGAACGGCATCTGTTACCCGGGCGACTTCATTGACTACGAGGAGCTCCGCGAACAGCTGTCTTCTGTCTCTTCTTTCGAACGTTTCGAAATTTTCCCAAAGGAATCCTCCTGGCCAAACCATAACACCAACGGAGTGACCGCCGCCTGTAGCCATGAGGGCAAGTCTTCTTTCTACCGTAATCTGCTGTGGCTGACTGAAAAAGAGGGTTCTTATCCCAAACTGAAGAACTCTTATGTCAACAAGAAGGGCAAAGAGGTCCTGGTGCTGTGGGGTATCCACCACCCCCCCAACTCCAAGGAGCAGCAGAATCTGTACCAAAACGAAAATGCTTACGTGTCTGTCGTGACATCTAACTACAACCGGCGCTTCACGCCCGAAATCGCCGAGCGTCCCAAAGTGCGCGATCAGGCCGGAAGGATGAACTACTACTGGACCCTGCTCAAACCCGGAGATACCATCATATTCGAAGCCAATGGCAATCTCATCGCTCCCATGTATGCTTTCGCTCTCTCTCGCGGATTCGGTTCCGGCATAATCACTAGCCAAGCCTCCATGCACGAGTGCAATACCAAGTGTCAAACTCCACTGGGAGCCATTCAAAGCTCCCTGCCCTACCAAAACATCCATCCTGTGACCATTGGAGAGTGCCCTAAATACGTGCGCAGCGCGAAACTGCGCATGGTGACCGGTCTCCGCAATATACCCTCCATACAATCGCGCGGCCTGTTTGGTGCTATCGCTGGCTTTATCGAGGGAGGATGGACTGGAATGATCGACGGCTGGTATGGCTATCATCATCAAAACGAACAGGGTTCCGGCTACGCCGCTGACCAAAAGTCCACTCAAAACGCCATTAACGGTATTACAAACAAAGTAAACACCGTGATAGAGAAAATGAATATCCAATTCACTGCCGTGGGCAAAGAGTTTAACAAGCTGGAGAAGCGCATGGAAAATCTGAACAAAAAAGTCGATGATGGCTTCCTCGACATCTGGACTTACAACGCCGAACTCCTCGTGCTGCTCGAAAACGAGAGGACTCTGGACTTCCACGACTCCAACGTGAAGAACCTGTACGAGAAAGTCAAATCCCAACTCAAGAACAACGCCAAAGAAATCGGCAACGGCTGCTTCGAGTTCTACCACAAATGTGATAACGAGTGTATGGAATCCGTACGGCAAGGCACTTACGACTACCCCAAATACAGCGAAGAGAGCAAATTGAACCGCGAGAAAGTGGACGGCGTGAAACTGGAGTCCATGGGCATCTATCAGCACCACCACCACCACCACTAATAG ngSTF2.HA1-1SEQ ID NO: 119ATGAAATTCTTAGTCAACGTTGCCCTTGTTTTTATGGTCGTATACATTTCTTACATCTATGCGGCCCAAGTGATCAACACCAACTCCCTGTCCCTGCTCACTCAAAACAACCTCCAGAAATCTCAGTCTGCTCTCGGTACTGCTATCGAGCGTCTCTCCTCCGGCCTGAGGATCAACTCCGCTAAAGATGACGCCGCCGGACAAGCTATCGCTAACCGCTTTACTGCCAACATTAAAGGCCTGACCCAAGCCTCTAGGAACGCCAATGATGGCATTTCTATCGCTCAAACCACCGAAGGCGCCCTGAACGAAATCAATAATAACCTGCAACGTGTCCGCGAACTCGCCGTCCAGTCCGCCCAATCTACCAACTCTCAGTCTGACCTGGACTCTATCCAAGCTGAGATCACCCAAAGGCTCAACGAGATCGATCGTGTGTCTGGTCAAACTCAGTTTAACGGCGTCAAAGTGCTGGCTCAAGACAACACACTCACCATCCAAGTAGGAGCCAATGACGGCGAGACAATCGATATCGACCTGAAGCAGATCAATTCTCAAACTCTGGGCCTGGACTCCCTGAACGTCCAAAAGGCTTACGACGTGAAGGACACCGCTGTGACAACCAAAGCCTATGCTAATCAAGGAACAACCCTGGACGTGTCCGGACTCGATGACGCCGCTATCAAGGCCGCTACTGGCGGCACTCAGGGAACTGCCTCTGTGACCGGAGGCGCTGTGAAGTTCGACGCCGATAACAACAAATACTTCGTCACTATTGGTGGTTTCACTGGCGCTGACGCCGCTAAGAACGGTGACTACGAGGTCAACGTCGCCACTGACGGAACAGTGACACTGGCCGCTGGCGCTACCAAGACCACAATGCCTGCTGGTGCTACTACTAAGACAGAGGTGCAGGAACTCAAGGACACCCCTGCCGTGGTGTCCGCCGATGCTAAGAACGCTCTCATTGCTGGTGGTGTCGACGCTACGGACGCCAACGGAGCCGAGCTTGTGAAAATGTCCTACACCGACAAGAACGGAAAGACTATCGAGGGAGGTTACGCTCTGAAGGCTGGCGATAAGTACTACGCCGCTGATTATGACGAAGCTACAGGCGCCATTAAAGCTAAAACCACATCTTATACCGCTGCCGATGGTACTACCAAGACTGCTGCCAATCAGCTGGGTGGAGTCGATGGAAAAACCGAAGTGGTCACAATCGACGGAAAGACCTATCAAGCCTCCAAGGCTGCTGGCCACGACTTCAAGGCCCAACCCGAACTGGCCGAGGCTGCTGCTAAAACCACTGAAAACCCCCTGCAGAAAATTGACGCTGCCCTGGCCCAAGTGGATGCCCTCCGCTCCGACCTCGGCGCTGTGCAAAACCGCTTCAACTCCGCTATTACAAACCTCGGCAATACCGTCAATCAGCTCTCTGAAGCTAGGTCTCGTATTGAGGATTCCGATTACGCTACCGAGGTCTCCCAGATGTCCCGTGCCCAAATTCTCCAACAAGCCGGCACCTCCGTCCTCGCCCAAGCCAATCAGGTGCCACAGAATGTGCTGAGCCTGCTGGCTTCTGGATCTGGATCTGGTTCTTCCCATAATGGCAAATTGTGTCGGCTGAAAGGCATCGCCCCTCTGCAACTCGGCAAGTGCAATATTGCGGGGTGGCTGTTGGGCAACCCAGAATGTGACCCCCTCCTGCCCGTCCGTTCTTGGTCTTACATCGTGGAAACACCTAACTCCGAGAACGGCATCTGTTACCCGGGCGACTTCATTGACTACGAGGAGCTCCGCGAACAGCTGTCTTCTGTCTCTTCTTTCGAACGTTTCGAAATTTTCCCAAAGGAATCCTCCTGGCCAAACCATAACACCAACGGAGTGACCGCCGCCTGTAGCCATGAGGGCAAGTCTTCTTTCTACCGTAATCTGCTGTGGCTGACTGAAAAAGAGGGTTCTTATCCCAAACTGAAGAACTCTTATGTCAACAAGAAGGGCAAAGAGGTCCTGGTGCTGTGGGGTATCCACCACCCCCCCAACTCCAAGGAGCAGCAGAATCTGTACCAAAACGAAAATGCTTACGTGTCTGTCGTGACATCTAACTACAACCGGCGCTTCACGCCCGAAATCGCCGAGCGTCCCAAAGTGCGCGATCAGGCCGGAAGGATGAACTACTACTGGACCCTGCTCAAACCCGGAGATACCATCATATTCGAAGCCAATGGCAATCTCATCGCTCCCATGTATGCTTTCGCTCTCTCTCGCGGATTCGGTTCCGGCATAATCACTAGCCAAGCCTCCATGCACGAGTGCAATACCAAGTGTCAAACTCCACTGGGAGCCATTCAAAGCTCCCTGCCCTACCAAAACATCCATCCTGTGACCATTGGAGAGTGCCCTAAATACGTGCGCCACCACCACCACCACCACTAATAG ng HA1-1 SEQ ID NO: 120TCCCATAATGGCAAATTGTGTCGGCTGAAAGGCATCGCCCCTCTGCAACTCGGCAAGTGCAATATTGCGGGGTGGCTGTTGGGCAACCCAGAATGTGACCCCCTCCTGCCCGTCCGTTCTTGGTCTTACATCGTGGAAACACCTAACTCCGAGAACGGCATCTGTTACCCGGGCGACTTCATTGACTACGAGGAGCTCCGCGAACAGCTGTCTTCTGTCTCTTCTTTCGAACGTTTCGAAATTTTCCCAAAGGAATCCTCCTGGCCAAACCATAACACCAACGGAGTGACCGCCGCCTGTAGCCATGAGGGCAAGTCTTCTTTCTACCGTAATCTGCTGTGGCTGACTGAAAAAGAGGGTTCTTATCCCAAACTGAAGAACTCTTATGTCAACAAGAAGGGCAAAGAGGTCCTGGTGCTGTGGGGTATCCACCACCCCCCCAACTCCAAGGAGCAGCAGAATCTGTACCAAAACGAAAATGCTTACGTGTCTGTCGTGACATCTAACTACAACCGGCGCTTCACGCCCGAAATCGCCGAGCGTCCCAAAGTGCGCGATCAGGCCGGAAGGATGAACTACTACTGGACCCTGCTCAAACCCGGAGATACCATCATATTCGAAGCCAATGGCAATCTCATCGCTCCCATGTATGCTTTCGCTCTCTCTCGCGGATTCGGTTCCGGCATAATCACTAGCCAAGCCTCCATGCACGAGTGCAATACCAAGTGTCAAACTCCACTGGGAGCCATTCAAAGCTCCCTGCCCTACCAAAACATCCATCCTGTGACCATTGGAGAGTGCCCTAAATACGTGCGCCACCACCACCACCACCACTAATAGngSTF2.HA1-2 SEQ ID NO: 121ATGAAATTCTTAGTCAACGTTGCCCTTGTTTTTATGGTCGTATACATTTCTTACATCTATGCGGCCCAAGTGATCAACACCAACTCCCTGTCCCTGCTCACTCAAAACAACCTCCAGAAATCTCAGTCTGCTCTCGGTACTGCTATCGAGCGTCTCTCCTCCGGCCTGAGGATCAACTCCGCTAAAGATGACGCCGCCGGACAAGCTATCGCTAACCGCTTTACTGCCAACATTAAAGGCCTGACCCAAGCCTCTAGGAACGCCAATGATGGCATTTCTATCGCTCAAACCACCGAAGGCGCCCTGAACGAAATCAATAATAACCTGCAACGTGTCCGCGAACTCGCCGTCCAGTCCGCCCAATCTACCAACTCTCAGTCTGACCTGGACTCTATCCAAGCTGAGATCACCCAAAGGCTCAACGAGATCGATCGTGTGTCTGGTCAAACTCAGTTTAACGGCGTCAAAGTGCTGGCTCAAGACAACACACTCACCATCCAAGTAGGAGCCAATGACGGCGAGACAATCGATATCGACCTGAAGCAGATCAATTCTCAAACTCTGGGCCTGGACTCCCTGAACGTCCAAAAGGCTTACGACGTGAAGGACACCGCTGTGACAACCAAAGCCTATGCTAATCAAGGAACAACCCTGGACGTGTCCGGACTCGATGACGCCGCTATCAAGGCCGCTACTGGCGGCACTCAGGGAACTGCCTCTGTGACCGGAGGCGCTGTGAAGTTCGACGCCGATAACAACAAATACTTCGTCACTATTGGTGGTTTCACTGGCGCTGACGCCGCTAAGAACGGTGACTACGAGGTCAACGTCGCCACTGACGGAACAGTGACACTGGCCGCTGGCGCTACCAAGACCACAATGCCTGCTGGTGCTACTACTAAGACAGAGGTGCAGGAACTCAAGGACACCCCTGCCGTGGTGTCCGCCGATGCTAAGAACGCTCTCATTGCTGGTGGTGTCGACGCTACGGACGCCAACGGAGCCGAGCTTGTGAAAATGTCCTACACCGACAAGAACGGAAAGACTATCGAGGGAGGTTACGCTCTGAAGGCTGGCGATAAGTACTACGCCGCTGATTATGACGAAGCTACAGGCGCCATTAAAGCTAAAACCACATCTTATACCGCTGCCGATGGTACTACCAAGACTGCTGCCAATCAGCTGGGTGGAGTCGATGGAAAAACCGAAGTGGTCACAATCGACGGAAAGACCTATCAAGCCTCCAAGGCTGCTGGCCACGACTTCAAGGCCCAACCCGAACTGGCCGAGGCTGCTGCTAAAACCACTGAAAACCCCCTGCAGAAAATTGACGCTGCCCTGGCCCAAGTGGATGCCCTCCGCTCCGACCTCGGCGCTGTGCAAAACCGCTTCAACTCCGCTATTACAAACCTCGGCAATACCGTCAATCAGCTCTCTGAAGCTAGGTCTCGTATTGAGGATTCCGATTACGCTACCGAGGTCTCCCAGATGTCCCGTGCCCAAATTCTCCAACAAGCCGGCACCTCCGTCCTCGCCCAAGCCAATCAGGTGCCACAGAATGTGCTGAGCCTACTAGCATCAGGTTCCGGCTCAGGTTCCAAAGGCATAGCGCCACTGCAGCTCGGCAAATGTAACATCGCAGGTTGGCTCCTTGGTAACCCGGAGTGCGACCCCCTCCTCCCTGTACGATCCTGGAGTTATATCGTGGAGACCCCCAATAGCGAGAACGGAATTTGCTACCCAGGAGATTTCATAGACTACGAGGAGTTGCGCGAGCAGCTTTCGTCTGTGAGCAGCTTCGAGAGGTTCGAAATCTTCCCGAAGGAGAGCAGCTGGCCGAATCATAACACTAACGGTGTGACAGCCGCCTGCAGTCATGAAGGAAAGAGTTCATTCTATCGCAACCTGCTGTGGTTGACGGAGAAAGAGGGCAGCTACCCTAAGTTGAAGAACTCCTATGTGAACAAAAAAGGCAAGGAGGTTCTGGTGCTGTGGGGCATACACCACCCCCCCAATAGCAAGGAGCAGCAGAATCTGTACCAAAACGAGAATGCCTATGTGAGCGTGGTCACTAGTAACTATAACCGTCGGTTCACTCCCGAGATCGCCGAGCGTCCGAAGGTGAGGGACCAGGCAGGCCGGATGAACTACTACTGGACCCTATTGAAGCCAGGGGACACGATTATCTTCGAGGCAAACGGAAACCTCATAGCGCCGATGTACGCGTTCGCCCTGAGCCGTGGCTTTGGATCGGGGATCATCACGTCTCACCATCATCACCACCATTAATAG ng HA1-2 SEQ ID NO: 122AAAGGCATAGCGCCACTGCAGCTCGGCAAATGTAACATCGCAGGTTGGCTCCTTGGTAACCCGGAGTGCGACCCCCTCCTCCCTGTACGATCCTGGAGTTATATCGTGGAGACCCCCAATAGCGAGAACGGAATTTGCTACCCAGGAGATTTCATAGACTACGAGGAGTTGCGCGAGCAGCTTTCGTCTGTGAGCAGCTTCGAAAGGTTCGAGATATTCCCGAAGGAGAGCAGCTGGCCGAATCATAACACTAACGGTGTGACAGCCGCCTGCAGTCATGAAGGAAAGAGTTCATTCTATCGCAACCTGCTGTGGTTGACGGAGAAAGAGGGCAGCTACCCTAAGTTGAAGAACTCCTATGTGAACAAAAAAGGCAAGGAGGTTCTGGTGCTGTGGGGCATACACCACCCCCCCAATAGCAAGGAGCAGCAGAATCTGTACCAAAACGAGAATGCCTATGTGAGCGTGGTCACTAGTAACTATAACCGTCGGTTCACTCCCGAGATCGCCGAGCGTCCGAAGGTGAGGGACCAGGCAGGCCGGATGAACTACTACTGGACCCTATTGAAGCCAGGGGACACGATTATCTTCGAGGCAAACGGAAACCTCATAGCGCCGATGTACGCCTTCGCCCTGAGCCGTGGCTTTGGATCGGGGATCATCACGTCTCACCATCATCACCACCATTAATAG ngSTF2.HA1-2mut SEQ ID NO: 123ATGAAATTCTTAGTCAACGTTGCCCTTGTTTTTATGGTCGTATACATTTCTTACATCTATGCGGCCCAAGTGATCAACACCAACTCCCTGTCCCTGCTCACTCAAAACAACCTCCAGAAATCTCAGTCTGCTCTCGGTACTGCTATCGAGCGTCTCTCCTCCGGCCTGAGGATCAACTCCGCTAAAGATGACGCCGCCGGACAAGCTATCGCTAACCGCTTTACTGCCAACATTAAAGGCCTGACCCAAGCCTCTAGGAACGCCAATGATGGCATTTCTATCGCTCAAACCACCGAAGGCGCCCTGAACGAAATCAATAATAACCTGCAACGTGTCCGCGAACTCGCCGTCCAGTCCGCCCAATCTACCAACTCTCAGTCTGACCTGGACTCTATCCAAGCTGAGATCACCCAAAGGCTCAACGAGATCGATCGTGTGTCTGGTCAAACTCAGTTTAACGGCGTCAAAGTGCTGGCTCAAGACAACACACTCACCATCCAAGTAGGAGCCAATGACGGCGAGACAATCGATATCGACCTGAAGCAGATCAATTCTCAAACTCTGGGCCTGGACTCCCTGAACGTCCAAAAGGCTTACGACGTGAAGGACACCGCTGTGACAACCAAAGCCTATGCTAATCAAGGAACAACCCTGGACGTGTCCGGACTCGATGACGCCGCTATCAAGGCCGCTACTGGCGGCACTCAGGGAACTGCCTCTGTGACCGGAGGCGCTGTGAAGTTCGACGCCGATAACAACAAATACTTCGTCACTATTGGTGGTTTCACTGGCGCTGACGCCGCTAAGAACGGTGACTACGAGGTCAACGTCGCCACTGACGGAACAGTGACACTGGCCGCTGGCGCTACCAAGACCACAATGCCTGCTGGTGCTACTACTAAGACAGAGGTGCAGGAACTCAAGGACACCCCTGCCGTGGTGTCCGCCGATGCTAAGAACGCTCTCATTGCTGGTGGTGTCGACGCTACGGACGCCAACGGAGCCGAGCTTGTGAAAATGTCCTACACCGACAAGAACGGAAAGACTATCGAGGGAGGTTACGCTCTGAAGGCTGGCGATAAGTACTACGCCGCTGATTATGACGAAGCTACAGGCGCCATTAAAGCTAAAACCACATCTTATACCGCTGCCGATGGTACTACCAAGACTGCTGCCAATCAGCTGGGTGGAGTCGATGGAAAAACCGAAGTGGTCACAATCGACGGAAAGACCTATCAAGCCTCCAAGGCTGCTGGCCACGACTTCAAGGCCCAACCCGAACTGGCCGAGGCTGCTGCTAAAACCACTGAAAACCCCCTGCAGAAAATTGACGCTGCCCTGGCCCAAGTGGATGCCCTCCGCTCCGACCTCGGCGCTGTGCAAAACCGCTTCAACTCCGCTATTACAAACCTCGGCAATACCGTCAATCAGCTCTCTGAAGCTAGGTCTCGTATTGAGGATTCCGATTACGCTACCGAGGTCTCCCAGATGTCCCGTGCCCAAATTCTCCAACAAGCCGGCACCTCCGTCCTCGCCCAAGCCAATCAGGTGCCACAGAATGTGCTGAGCCTGCTGGCTTCTGGATCTGGATCTGGTTCTAAAGGCGCTGCCCCTCTGCAACTCGGCAAGTGCAATATTGCGGGGTGGCTGTTGGGCAACCCAGAATGTGACCCCCTCCTGCCCGTCCGTTCTTGGTCTGACATCGCTGAAACACCTAACTCCGAGAACGGCATCTGTTACCCGGGCGACTTCATTGACTACGAGGAGCTCCGCGAACAGCTGTCTTCTGTCTCTTCTTTCGAACGTTTCGAAATTTTCCCAAAGGAATCCTCCTGGCCAAACCATAACACCAACGGAGTGACCGCCGCCTGTAGCCATGAGGGCAAGTCTTCTTTCTACCGTAATCTGCTGTGGCTGACTGAAAAAGAGGGTTCTTATCCCAAACTGAAGAACTCTTATGTCAACAAGAAGGGCAAAGAGGTCCTGGTGCTGTGGGGTATCCACCACCCCCCCAACTCCAAGGAGCAGCAGAATCTGTACCAAAACGAAAATGCTTACGTGTCTGTCGTGACATCTAACTACAACCGGCGCTTCACGCCCGAAATCGCCGAGCGTCCCAAAGTGCGCGATCAGGCCGGAAGGATGAACTACTACTGGACCCTGCTCAAACCCGGAGATACCATCATATTCGAAGCCAATGGCAATCTCATCGCTCCCATGTATGCTTTCGCTCTCTCTCGCGGATTCGGTTCCGGCATAATCACTAGCCACCACCACCACCACCACTAATAG ngHA1-2mut SEQ ID NO: 124AAAGGCGCTGCCCCTCTGCAACTCGGCAAGTGCAATATTGCGGGGTGGCTGTTGGGCAACCCAGAATGTGACCCCCTCCTGCCCGTCCGTTCTTGGTCTGACATCGCTGAAACACCTAACTCCGAGAACGGCATCTGTTACCCGGGCGACTTCATTGACTACGAGGAGCTCCGCGAACAGCTGTCTTCTGTCTCTTCTTTCGAACGTTTCGAAATTTTCCCAAAGGAATCCTCCTGGCCAAACCATAACACCAACGGAGTGACCGCCGCCTGTAGCCATGAGGGCAAGTCTTCTTTCTACCGTAATCTGCTGTGGCTGACTGAAAAAGAGGGTTCTTATCCCAAACTGAAGAACTCTTATGTCAACAAGAAGGGCAAAGAGGTCCTGGTGCTGTGGGGTATCCACCACCCCCCCAACTCCAAGGAGCAGCAGAATCTGTACCAAAACGAAAATGCTTACGTGTCTGTCGTGACATCTAACTACAACCGGCGCTTCACGCCCGAAATCGCCGAGCGTCCCAAAGTGCGCGATCAGGCCGGAAGGATGAACTACTACTGGACCCTGCTCAAACCCGGAGATACCATCATATTCGAAGCCAATGGCAATCTCATCGCTCCCATGTATGCTTTCGCTCTCTCTCGCGGATTCGGTTCCGGCATAATCACTAGCCACCACCACCACCACCACTAATAG ngSTF2.HA1-3 SEQ ID NO: 125ATGAAATTCTTAGTCAACGTTGCCCTTGTTTTTATGGTCGTATACATTTCTTACATCTATGCGGCCCAAGTGATCAACACCAACTCCCTGTCCCTGCTCACTCAAAACAACCTCCAGAAATCTCAGTCTGCTCTCGGTACTGCTATCGAGCGTCTCTCCTCCGGCCTGAGGATCAACTCCGCTAAAGATGACGCCGCCGGACAAGCTATCGCTAACCGCTTTACTGCCAACATTAAAGGCCTGACCCAAGCCTCTAGGAACGCCAATGATGGCATTTCTATCGCTCAAACCACCGAAGGCGCCCTGAACGAAATCAATAATAACCTGCAACGTGTCCGCGAACTCGCCGTCCAGTCCGCCCAATCTACCAACTCTCAGTCTGACCTGGACTCTATCCAAGCTGAGATCACCCAAAGGCTCAACGAGATCGATCGTGTGTCTGGTCAAACTCAGTTTAACGGCGTCAAAGTGCTGGCTCAAGACAACACACTCACCATCCAAGTAGGAGCCAATGACGGCGAGACAATCGATATCGACCTGAAGCAGATCAATTCTCAAACTCTGGGCCTGGACTCCCTGAACGTCCAAAAGGCTTACGACGTGAAGGACACCGCTGTGACAACCAAAGCCTATGCTAATCAAGGAACAACCCTGGACGTGTCCGGACTCGATGACGCCGCTATCAAGGCCGCTACTGGCGGCACTCAGGGAACTGCCTCTGTGACCGGAGGCGCTGTGAAGTTCGACGCCGATAACAACAAATACTTCGTCACTATTGGTGGTTTCACTGGCGCTGACGCCGCTAAGAACGGTGACTACGAGGTCAACGTCGCCACTGACGGAACAGTGACACTGGCCGCTGGCGCTACCAAGACCACAATGCCTGCTGGTGCTACTACTAAGACAGAGGTGCAGGAACTCAAGGACACCCCTGCCGTGGTGTCCGCCGATGCTAAGAACGCTCTCATTGCTGGTGGTGTCGACGCTACGGACGCCAACGGAGCCGAGCTTGTGAAAATGTCCTACACCGACAAGAACGGAAAGACTATCGAGGGAGGTTACGCTCTGAAGGCTGGCGATAAGTACTACGCCGCTGATTATGACGAAGCTACAGGCGCCATTAAAGCTAAAACCACATCTTATACCGCTGCCGATGGTACTACCAAGACTGCTGCCAATCAGCTGGGTGGAGTCGATGGAAAAACCGAAGTGGTCACAATCGACGGAAAGACCTATCAAGCCTCCAAGGCTGCTGGCCACGACTTCAAGGCCCAACCCGAACTGGCCGAGGCTGCTGCTAAAACCACTGAAAACCCCCTGCAGAAAATTGACGCTGCCCTGGCCCAAGTGGATGCCCTCCGCTCCGACCTCGGCGCTGTGCAAAACCGCTTCAACTCCGCTATTACAAACCTCGGCAATACCGTCAATCAGCTCTCTGAAGCTAGGTCTCGTATTGAGGATTCCGATTACGCTACCGAGGTCTCCCAGATGTCCCGTGCCCAAATTCTCCAACAAGCCGGCACCTCCGTCCTCGCCCAAGCCAATCAGGTGCCACAGAATGTGCTGAGCCTACTAGCATCAGGTTCCGGCTCAGGTTCCAATAGCGAGAACGGAATTTGCTACCCAGGAGATTTCATAGACTACGAGGAGTTGCGCGAGCAGCTTTCGTCTGTGAGCAGCTTCGAGAGGTTCGAAATCTTCCCGAAGGAGAGCAGCTGGCCGAATCATAACACTAACGGTGTGACAGCCGCCTGCAGTCATGAAGGAAAGAGTTCATTCTATCGCAACCTGCTGTGGTTGACGGAGAAGGAGGGCAGCTACCCTAAGCTGAAGAACTCCTATGTGAACAAAAAAGGCAAGGAGGTTCTGGTGCTGTGGGGCATACACCACCCCCCCAATAGCAAGGAGCAGCAGAATCTGTACCAAAACGAGAATGCCTATGTGAGCGTGGTCACTAGTAACTATAACCGTCGGTTCACTCCCGAGATCGCCGAGCGTCCGAAGGTGAGGGACCAGGCAGGCCGGATGAACTACTACTGGACCCTATTGAAGCCAGGGGACACGATTATCTTCGAGGCAAACGGAAACCTCATAGCGCCGATGTACGCCTTCGCCCTGAGCCGTGGCCACCATCATCACCACCATTAATAG ngHA1-3SEQ ID NO: 126AATAGCGAGAACGGAATTTGCTACCCAGGAGATTTCATAGACTACGAGGAGTTGCGCGAGCAGCTTTCGTCTGTGAGCAGCTTCGAAAGGTTCGAGATATTCCCGAAGGAGAGCAGCTGGCCGAATCATAACACTAACGGTGTGACAGCCGCCTGCAGTCATGAAGGAAAGAGTTCATTCTATCGCAACCTGCTGTGGTTGACGGAGAAAGAGGGCAGCTACCCTAAGTTGAAGAACTCCTATGTGAACAAAAAAGGCAAGGAGGTTCTGGTGCTGTGGGGCATACACCACCCCCCCAATAGCAAGGAGCAGCAGAATCTGTACCAAAACGAGAATGCCTATGTGAGCGTGGTCACTAGTAACTATAACCGTCGGTTCACTCCCGAGATCGCCGAGCGTCCGAAGGTGAGGGACCAGGCAGGCCGGATGAACTACTACTGGACCCTATTGAAGCCAGGGGACACGATTATCTTCGAGGCAAACGGAAACCTCATAGCGCCGATGTACGCCTTCGCCCTGAGCCGTGGCCACCATCATCACCACCATTAATAG ngSTF2.HA1-3mut SEQ ID NO: 127ATGAAATTCTTAGTCAACGTTGCCCTTGTTTTTATGGTCGTATACATTTCTTACATCTATGCGGCCCAAGTGATCAACACCAACTCCCTGTCCCTGCTCACTCAAAACAACCTCCAGAAATCTCAGTCTGCTCTCGGTACTGCTATCGAGCGTCTCTCCTCCGGCCTGAGGATCAACTCCGCTAAAGATGACGCCGCCGGACAAGCTATCGCTAACCGCTTTACTGCCAACATTAAAGGCCTGACCCAAGCCTCTAGGAACGCCAATGATGGCATTTCTATCGCTCAAACCACCGAAGGCGCCCTGAACGAAATCAATAATAACCTGCAACGTGTCCGCGAACTCGCCGTCCAGTCCGCCCAATCTACCAACTCTCAGTCTGACCTGGACTCTATCCAAGCTGAGATCACCCAAAGGCTCAACGAGATCGATCGTGTGTCTGGTCAAACTCAGTTTAACGGCGTCAAAGTGCTGGCTCAAGACAACACACTCACCATCCAAGTAGGAGCCAATGACGGCGAGACAATCGATATCGACCTGAAGCAGATCAATTCTCAAACTCTGGGCCTGGACTCCCTGAACGTCCAAAAGGCTTACGACGTGAAGGACACCGCTGTGACAACCAAAGCCTATGCTAATCAAGGAACAACCCTGGACGTGTCCGGACTCGATGACGCCGCTATCAAGGCCGCTACTGGCGGCACTCAGGGAACTGCCTCTGTGACCGGAGGCGCTGTGAAGTTCGACGCCGATAACAACAAATACTTCGTCACTATTGGTGGTTTCACTGGCGCTGACGCCGCTAAGAACGGTGACTACGAGGTCAACGTCGCCACTGACGGAACAGTGACACTGGCCGCTGGCGCTACCAAGACCACAATGCCTGCTGGTGCTACTACTAAGACAGAGGTGCAGGAACTCAAGGACACCCCTGCCGTGGTGTCCGCCGATGCTAAGAACGCTCTCATTGCTGGTGGTGTCGACGCTACGGACGCCAACGGAGCCGAGCTTGTGAAAATGTCCTACACCGACAAGAACGGAAAGACTATCGAGGGAGGTTACGCTCTGAAGGCTGGCGATAAGTACTACGCCGCTGATTATGACGAAGCTACAGGCGCCATTAAAGCTAAAACCACATCTTATACCGCTGCCGATGGTACTACCAAGACTGCTGCCAATCAGCTGGGTGGAGTCGATGGAAAAACCGAAGTGGTCACAATCGACGGAAAGACCTATCAAGCCTCCAAGGCTGCTGGCCACGACTTCAAGGCCCAACCCGAACTGGCCGAGGCTGCTGCTAAAACCACTGAAAACCCCCTGCAGAAAATTGACGCTGCCCTGGCCCAAGTGGATGCCCTCCGCTCCGACCTCGGCGCTGTGCAAAACCGCTTCAACTCCGCTATTACAAACCTCGGCAATACCGTCAATCAGCTCTCTGAAGCTAGGTCTCGTATTGAGGATTCCGATTACGCTACCGAGGTCTCCCAGATGTCCCGTGCCCAAATTCTCCAACAAGCCGGCACCTCCGTCCTCGCCCAAGCCAATCAGGTGCCACAGAATGTGCTGAGCCTGCTGGCTTCTGGATCTGGATCTGGTTCTAACTCCGAGAACGAAATCTGTTACCCGGGCGACTTCATTGACAAAGAGGAGCTCCGCGAACAGCTGTCTTCTGTCTCTTCTTTCGAACGTTTCGAAATTTTCCCAAAGGAATCCTCCTGGCCAAACCATAACACCAACGGAGTGACCGCCGCCTGTAGCCATGAGGGCAAGTCTTCTTTCTACCGTAATCTGCTGTGGCTGACTGAAAAAGAGGGTTCTTATCCCAAACTGAAGAACTCTTATGTCAACAAGAAGGGCAAAGAGGTCCTGGTGCTGTGGGGTATCCACCACCCCCCCAACTCCAAGGAGCAGCAGAATCTGTACCAAAACGAAAATGCTTACGTGTCTGTCGTGACATCTAACTACAACCGGCGCTTCACGCCCGAAATCGCCGAGCGTCCCAAAGTGCGCGATCAGGCCGGAAGGATGAACTACTACTGGACCCTGCTCAAACCCGGAGATACCATCATATTCGAAGCCAATGGCAATCTCATCGCTCCCATGTATGCTGCTGCTCTCTCTCGCGGACACCACCACCACCACCACTAATAG ngHA1-3mutSEQ ID NO: 128AACTCCGAGAACGAAATCTGTTACCCGGGCGACTTCATTGACAAAGAGGAGCTCCGCGAACAGCTGTCTTCTGTCTCTTCTTTCGAACGTTTCGAAATTTTCCCAAAGGAATCCTCCTGGCCAAACCATAACACCAACGGAGTGACCGCCGCCTGTAGCCATGAGGGCAAGTCTTCTTTCTACCGTAATCTGCTGTGGCTGACTGAAAAAGAGGGTTCTTATCCCAAACTGAAGAACTCTTATGTCAACAAGAAGGGCAAAGAGGTCCTGGTGCTGTGGGGTATCCACCACCCCCCCAACTCCAAGGAGCAGCAGAATCTGTACCAAAACGAAAATGCTTACGTGTCTGTCGTGACATCTAACTACAACCGGCGCTTCACGCCCGAAATCGCCGAGCGTCCCAAAGTGCGCGATCAGGCCGGAAGGATGAACTACTACTGGACCCTGCTCAAACCCGGAGATACCATCATATTCGAAGCCAATGGCAATCTCATCGCTCCCATGTATGCTGCTGCTCTCTCTCGCGGACACCACCACCACCACCACTAATAG wtSTF2.HA1-1ng SEQ ID NO: 129ATGAAATTCTTAGTCAACGTTGCCCTTGTTTTTATGGTCGTATACATTTCTTACATCTATGCGGCCCAGGTTATCAATACCAACTCCCTGTCGTTGCTCACCCAAAATAACCTTAATAAAAGCCAGAGCGCACTGGGAACCGCCATAGAACGCCTCTCAAGCGGCCTCCGGATCAATTCTGCAAAAGACGACGCCGCCGGTCAGGCCATCGCAAACCGCTTTACCGCCAATATCAAGGGACTGACGCAGGCTTCGAGGAATGCTAACGATGGAATAAGCATCGCTCAAACCACGGAGGGCGCCCTGAACGAGATCAACAACAACCTACAGCGCGTCAGGGAGCTCGCAGTGCAGTCCGCCAATTCGACCAACTCGCAGTCGGACCTGGACTCGATCCAAGCCGAAATCACCCAGCGCCTGAATGAGATTGACCGGGTGAGCGGTCAGACACAGTTTAACGGCGTGAAGGTACTTGCACAGGATAACACACTTACGATACAGGTGGGCGCCAACGATGGTGAAACCATAGACATTGATCTCAAACAGATTAACAGCCAGACGCTCGGGTTGGATAGCCTGAATGTGCAAAAGGCGTACGACGTGAAAGACACGGCGGTCACTACCAAAGCCTACGCTAACAATGGCACTACCTTGGATGTGAGCGGATTGGATGATGCAGCAATCAAGGCTGCTACCGGCGGTACGAACGGAACCGCGTCCGTGACCGGCGGTGCCGTGAAGTTCGATGCTGACAACAATAAGTATTTCGTCACCATTGGAGGCTTTACTGGCGCCGACGCAGCAAAGAACGGCGACTATGAAGTGAACGTGGCAACCGATGGAACCGTGACGCTGGCCGCTGGTGCCACCAAGACCACCATGCCAGCCGGCGCCACAACTAAGACCGAGGTGCAGGAGTTAAAGGACACCCCCGCGGTGGTTAGCGCAGATGCCAAAAACGCCTTGATCGCCGGCGGAGTGGATGCAACTGATGCTAATGGTGCGGAGCTGGTTAAAATGTCGTATACAGACAAGAATGGTAAGACGATCGAGGGCGGTTATGCCCTTAAGGCAGGAGATAAGTATTACGCTGCTGATTACGATGAGGCGACGGGAGCTATTAAGGCCAAGACAACGTCATACACGGCGGCGGACGGAACGACTAAGACGGCTGCCAATCAGTTGGGAGGGGTTGACGGGAAGACAGAGGTCGTTACGATCGATGGCAAGACATACAACGCTTCCAAGGCCGCTGGCCACGATTTCAAAGCTCAACCCGAACTGGCCGAGGCCGCGGCGAAAACAACTGAGAACCCGTTGCAGAAGATTGATGCGGCCCTGGCGCAAGTAGATGCCCTGCGCTCAGACCTGGGCGCCGTTCAAAATCGATTCAATTCCGCGATTACAAACCTGGGCAATACAGTAAACAATCTATCCGAGGCCAGATCCCGCATTGAAGACTCCGACTACGCGACAGAAGTAAGTAACATGAGTCGTGCCCAGATTCTGCAGCAGGCCGGCACTAGTGTCCTGGCCCAGGCCAATCAAGTCCCGCAGAATGTGCTGAGCCTACTAGCATCTGGATCTGGATCTGGTTCTTCCCATAATGGCAAATTGTGTCGGCTGAAAGGCATCGCCCCTCTGCAACTCGGCAAGTGCAATATTGCGGGGTGGCTGTTGGGCAACCCAGAATGTGACCCCCTCCTGCCCGTCCGTTCTTGGTCTTACATCGTGGAAACACCTAACTCCGAGAACGGCATCTGTTACCCGGGCGACTTCATTGACTACGAGGAGCTCCGCGAACAGCTGTCTTCTGTCTCTTCTTTCGAACGTTTCGAAATTTTCCCAAAGGAATCCTCCTGGCCAAACCATAACACCAACGGAGTGACCGCCGCCTGTAGCCATGAGGGCAAGTCTTCTTTCTACCGTAATCTGCTGTGGCTGACTGAAAAAGAGGGTTCTTATCCCAAACTGAAGAACTCTTATGTCAACAAGAAGGGCAAAGAGGTCCTGGTGCTGTGGGGTATCCACCACCCCCCCAACTCCAAGGAGCAGCAGAATCTGTACCAAAACGAAAATGCTTACGTGTCTGTCGTGACATCTAACTACAACCGGCGCTTCACGCCCGAAATCGCCGAGCGTCCCAAAGTGCGCGATCAGGCCGGAAGGATGAACTACTACTGGACCCTGCTCAAACCCGGAGATACCATCATATTCGAAGCCAATGGCAATCTCATCGCTCCCATGTATGCTTTCGCTCTCTCTCGCGGATTCGGTTCCGGCATAATCACTAGCCAAGCCTCCATGCACGAGTGCAATACCAAGTGTCAAACTCCACTGGGAGCCATTCAAAGCTCCCTGCCCTACCAAAACATCCATCCTGTGACCATTGGAGAGTGCCCTAAATACGTGCGCCACCACCACCACCACCACTAATAG ngHA1-1 SEQ ID NO: 130TCCCATAATGGCAAATTGTGTCGGCTGAAAGGCATCGCCCCTCTGCAACTCGGCAAGTGCAATATTGCGGGGTGGCTGTTGGGCAACCCAGAATGTGACCCCCTCCTGCCCGTCCGTTCTTGGTCTTACATCGTGGAAACACCTAACTCCGAGAACGGCATCTGTTACCCGGGCGACTTCATTGACTACGAGGAGCTCCGCGAACAGCTGTCTTCTGTCTCTTCTTTCGAACGTTTCGAAATTTTCCCAAAGGAATCCTCCTGGCCAAACCATAACACCAACGGAGTGACCGCCGCCTGTAGCCATGAGGGCAAGTCTTCTTTCTACCGTAATCTGCTGTGGCTGACTGAAAAAGAGGGTTCTTATCCCAAACTGAAGAACTCTTATGTCAACAAGAAGGGCAAAGAGGTCCTGGTGCTGTGGGGTATCCACCACCCCCCCAACTCCAAGGAGCAGCAGAATCTGTACCAAAACGAAAATGCTTACGTGTCTGTCGTGACATCTAACTACAACCGGCGCTTCACGCCCGAAATCGCCGAGCGTCCCAAAGTGCGCGATCAGGCCGGAAGGATGAACTACTACTGGACCCTGCTCAAACCCGGAGATACCATCATATTCGAAGCCAATGGCAATCTCATCGCTCCCATGTATGCTTTCGCTCTCTCTCGCGGATTCGGTTCCGGCATAATCACTAGCCAAGCCTCCATGCACGAGTGCAATACCAAGTGTCAAACTCCACTGGGAGCCATTCAAAGCTCCCTGCCCTACCAAAACATCCATCCTGTGACCATTGGAGAGTGCCCTAAATACGTGCGCCACCACCACCACCACCACTAATAGngSTF2.HA1-1wt SEQ ID NO: 131ATGAAATTCTTAGTCAACGTTGCCCTTGTTTTTATGGTCGTATACATTTCTTACATCTATGCGGCCCAAGTGATCAACACCAACTCCCTGTCCCTGCTCACTCAAAACAACCTCCAGAAATCTCAGTCTGCTCTCGGTACTGCTATCGAGCGTCTCTCCTCCGGCCTGAGGATCAACTCCGCTAAAGATGACGCCGCCGGACAAGCTATCGCTAACCGCTTTACTGCCAACATTAAAGGCCTGACCCAAGCCTCTAGGAACGCCAATGATGGCATTTCTATCGCTCAAACCACCGAAGGCGCCCTGAACGAAATCAATAATAACCTGCAACGTGTCCGCGAACTCGCCGTCCAGTCCGCCCAATCTACCAACTCTCAGTCTGACCTGGACTCTATCCAAGCTGAGATCACCCAAAGGCTCAACGAGATCGATCGTGTGTCTGGTCAAACTCAGTTTAACGGCGTCAAAGTGCTGGCTCAAGACAACACACTCACCATCCAAGTAGGAGCCAATGACGGCGAGACAATCGATATCGACCTGAAGCAGATCAATTCTCAAACTCTGGGCCTGGACTCCCTGAACGTCCAAAAGGCTTACGACGTGAAGGACACCGCTGTGACAACCAAAGCCTATGCTAATCAAGGAACAACCCTGGACGTGTCCGGACTCGATGACGCCGCTATCAAGGCCGCTACTGGCGGCACTCAGGGAACTGCCTCTGTGACCGGAGGCGCTGTGAAGTTCGACGCCGATAACAACAAATACTTCGTCACTATTGGTGGTTTCACTGGCGCTGACGCCGCTAAGAACGGTGACTACGAGGTCAACGTCGCCACTGACGGAACAGTGACACTGGCCGCTGGCGCTACCAAGACCACAATGCCTGCTGGTGCTACTACTAAGACAGAGGTGCAGGAACTCAAGGACACCCCTGCCGTGGTGTCCGCCGATGCTAAGAACGCTCTCATTGCTGGTGGTGTCGACGCTACGGACGCCAACGGAGCCGAGCTTGTGAAAATGTCCTACACCGACAAGAACGGAAAGACTATCGAGGGAGGTTACGCTCTGAAGGCTGGCGATAAGTACTACGCCGCTGATTATGACGAAGCTACAGGCGCCATTAAAGCTAAAACCACATCTTATACCGCTGCCGATGGTACTACCAAGACTGCTGCCAATCAGCTGGGTGGAGTCGATGGAAAAACCGAAGTGGTCACAATCGACGGAAAGACCTATCAAGCCTCCAAGGCTGCTGGCCACGACTTCAAGGCCCAACCCGAACTGGCCGAGGCTGCTGCTAAAACCACTGAAAACCCCCTGCAGAAAATTGACGCTGCCCTGGCCCAAGTGGATGCCCTCCGCTCCGACCTCGGCGCTGTGCAAAACCGCTTCAACTCCGCTATTACAAACCTCGGCAATACCGTCAATCAGCTCTCTGAAGCTAGGTCTCGTATTGAGGATTCCGATTACGCTACCGAGGTCTCCCAGATGTCCCGTGCCCAAATTCTCCAACAAGCCGGCACCTCCGTCCTCGCCCAAGCCAATCAGGTGCCACAGAATGTGCTGAGCCTACTAGCATCAGGTTCCGGCTCAGGTTCCAGCCACAACGGAAAGCTGTGCCGTCTGAAAGGCATAGCGCCACTGCAGCTCGGCAAATGTAACATCGCAGGTTGGCTCCTTGGTAACCCGGAGTGCGACCCCCTCCTCCCTGTACGATCCTGGAGTTATATCGTGGAGACCCCCAATAGCGAGAACGGAATTTGCTACCCAGGAGATTTCATAGACTACGAGGAGTTGCGCGAGCAGCTTTCGTCTGTGAGCAGCTTCGAGAGGTTCGAAATCTTCCCGAAGGAGAGCAGCTGGCCGAATCATAACACTAACGGTGTGACAGCCGCCTGCAGTCATGAAGGAAAGAGTTCATTCTATCGCAACCTGCTGTGGTTGACGGAGAAAGAGGGCAGCTACCCTAAGTTGAAGAACTCCTATGTGAACAAAAAAGGCAAGGAGGTTCTGGTGCTGTGGGGCATACACCACCCCCCCAATAGCAAGGAGCAGCAGAATCTGTACCAAAACGAGAATGCCTATGTGAGCGTGGTCACTAGTAACTATAACCGTCGGTTCACTCCCGAGATCGCCGAGCGTCCGAAGGTGAGGGACCAGGCAGGCCGGATGAACTACTACTGGACCCTATTGAAGCCAGGGGACACGATTATCTTCGAGGCAAACGGAAACCTCATAGCGCCGATGTACGCGTTCGCCCTGAGCCGCGGCTTTGGATCGGGGATCATCACGTCTAACGCCTCGATGCACGAATGTAATACCAAATGCCAGACCCCACTGGGTGCTATCAACTCGTCCTTACCCTATCAAAATATACATCCGGTCACCATAGGCGAGTGTCCCAAATATGTCAGACACCATCATCACCACCATTAATAG HA1-1 PR8 SEQ ID NO: 132AGCCACAACGGAAAGCTGTGCCGTCTGAAAGGCATAGCGCCACTGCAGCTCGGCAAATGTAACATCGCAGGTTGGCTCCTTGGTAACCCGGAGTGCGACCCCCTCCTCCCTGTACGATCCTGGAGTTATATCGTGGAGACCCCCAATAGCGAGAACGGAATTTGCTACCCAGGAGATTTCATAGACTACGAGGAGTTGCGCGAGCAGCTTTCGTCTGTGAGCAGCTTCGAAAGGTTCGAGATATTCCCGAAGGAGAGCAGCTGGCCGAATCATAACACTAACGGTGTGACAGCCGCCTGCAGTCATGAAGGAAAGAGTTCATTCTATCGCAACCTGCTGTGGTTGACGGAGAAAGAGGGCAGCTACCCTAAGTTGAAGAACTCCTATGTGAACAAAAAAGGCAAGGAGGTTCTGGTGCTGTGGGGCATACACCACCCCCCCAATAGCAAGGAGCAGCAGAATCTGTACCAAAACGAGAATGCCTATGTGAGCGTGGTCACTAGTAACTATAACCGTCGGTTCACTCCCGAGATCGCCGAGCGTCCGAAGGTGAGGGACCAGGCAGGCCGGATGAACTACTACTGGACCCTATTGAAGCCAGGGGACACGATTATCTTCGAGGCAAACGGAAACCTCATAGCGCCGATGTACGCCTTCGCCCTGAGCCGTGGCTTTGGATCGGGGATCATCACGTCTAACGCCTCGATGCACGAATGTAATACCAAATGCCAGACCCCACTGGGTGCTATCAACTCGTCCTTACCCTATCAAAATATACATCCGGTCACCATAGGCGAGTGTCCCAAATATGTCAGACACCATCATCACCACCATTAATAG HA1-1 PR8SEQ ID NO: 133ATGAAATTCTTAGTCAACGTTGCCCTTGTTTTTATGGTCGTATACATTTCTTACATCTATGCGAGCCACAACGGAAAGCTGTGCCGTCTGAAAGGCATAGCGCCACTGCAGCTCGGCAAATGTAACATCGCAGGTTGGCTCCTTGGTAACCCGGAGTGCGACCCCCTCCTCCCTGTACGATCCTGGAGTTATATCGTGGAGACCCCCAATAGCGAGAACGGAATTTGCTACCCAGGAGATTTCATAGACTACGAGGAGTTGCGCGAGCAGCTTTCGTCTGTGAGCAGCTTCGAAAGGTTCGAGATATTCCCGAAGGAGAGCAGCTGGCCGAATCATAACACTAACGGTGTGACAGCCGCCTGCAGTCATGAAGGAAAGAGTTCATTCTATCGCAACCTGCTGTGGTTGACGGAGAAAGAGGGCAGCTACCCTAAGTTGAAGAACTCCTATGTGAACAAAAAAGGCAAGGAGGTTCTGGTGCTGTGGGGCATACACCACCCCCCCAATAGCAAGGAGCAGCAGAATCTGTACCAAAACGAGAATGCCTATGTGAGCGTGGTCACTAGTAACTATAACCGTCGGTTCACTCCCGAGATCGCCGAGCGTCCGAAGGTGAGGGACCAGGCAGGCCGGATGAACTACTACTGGACCCTATTGAAGCCAGGGGACACGATTATCTTCGAGGCAAACGGAAACCTCATAGCGCCGATGTACGCCTTCGCCCTGAGCCGTGGCTTTGGATCGGGGATCATCACGTCTAACGCCTCGATGCACGAATGTAATACCAAATGCCAGACCCCACTGGGTGCTATCAACTCGTCCTTACCCTATCAAAATATACATCCGGTCACCATAGGCGAGTGTCCCAAATATGTCAGACACCATCATCACCACCATTAAT AGFOR PRIMER SEQ ID NO: 134AGGCAGATCTATGAAATTCTTAGTCAACGTTGCCCTTGTTTTTATGGTCGTATACATTTCTTACATCTATGCGAGCCACAACGGAAAGCTGTGCCGTCTGAAAGG REV PRIMER SEQ ID NO: 135ACCTGCATGCCTATTAATGGTGGTGATGATGGTGTCTGACATATTTGGGACACTC HA0s PR8SEQ ID NO: 136ATGAAATTCTTAGTCAACGTTGCCCTTGTTTTTATGGTCGTATACATTTCTTACATCTATGCGGACACCATTTGCATTGGATACCATGCAAACAACTCAACCGATACTGTTGATACCGTCCTTGAGAAGAACGTTACCGTCACGCACTCGGTCAACCTATTAGAGGATAGCCACAACGGAAAGCTGTGCCGTCTGAAAGGCATAGCGCCACTGCAGCTCGGCAAATGTAACATCGCAGGTTGGCTCCTTGGTAACCCGGAGTGCGACCCCCTCCTCCCTGTACGATCCTGGAGTTATATCGTGGAGACCCCCAATAGCGAGAACGGAATTTGCTACCCAGGAGATTTCATAGACTACGAGGAGTTGCGCGAGCAGCTTTCGTCTGTGAGCAGCTTCGAAAGGTTCGAGATATTCCCGAAGGAGAGCAGCTGGCCGAATCATAACACTAACGGTGTGACAGCCGCCTGCAGTCATGAAGGAAAGAGTTCATTCTATCGCAACCTGCTGTGGTTGACGGAGAAAGAGGGCAGCTACCCTAAGTTGAAGAACTCCTATGTGAACAAAAAAGGCAAGGAGGTTCTGGTGCTGTGGGGCATACACCACCCCCCCAATAGCAAGGAGCAGCAGAATCTGTACCAAAACGAGAATGCCTATGTGAGCGTGGTCACTAGTAACTATAACCGTCGGTTCACTCCCGAGATCGCCGAGCGTCCGAAGGTGAGGGACCAGGCAGGCCGGATGAACTACTACTGGACCCTATTGAAGCCAGGGGACACGATTATCTTCGAGGCAAACGGAAACCTCATAGCGCCGATGTACGCCTTCGCCCTGAGCCGTGGCTTTGGATCGGGGATCATCACGTCTAACGCCTCGATGCACGAATGTAATACCAAATGCCAGACCCCACTGGGTGCTATCAACTCGTCCTTACCCTATCAAAATATACATCCGGTCACCATAGGCGAGTGTCCCAAATATGTCAGATCCGCCAAGTTGCGGATGGTGACCGGCCTCCGCAATATTCCTAGTATTCAGTCACGCGGCTTGTTCGGCGCCATCGCTGGTTTCATCGAAGGCGGGTGGACAGGCATGATTGATGGCTGGTATGGCTATCACCACCAGAACGAGCAGGGCTCGGGCTACGCGGCTGACCAGAAGTCGACTCAGAATGCCATCAATGGCATCACGAATAAGGTGAACACGGTCATTGAAAAGATGAACATTCAATTTACAGCCGTAGGAAAAGAGTTTAATAAACTGGAAAAAAGAATGGAGAATCTGAATAAGAAGGTGGACGACGGATTTTTGGACATCTGGACGTACAACGCCGAGCTGCTGGTTCTGCTGGAAAATGAGCGAACACTGGATTTTCATGATTCTAACGTAAAGAATCTGTACGAGAAGGTGAAGTCCCAACTAAAGAATAATGCCAAGGAAATCGGAAATGGATGCTTTGAGTTTTACCACAAGTGCGATAATGAGTGCATGGAATCCGTGCGAAATGGTACATACGATTACCCAAAGTACTCCGAAGAATCCAAGCTAAATCGCGAAAAGGTTGATGGTGTTAAACTTGAATCCATGGGTATTTACCAACACCATCATCACCACCATTAATAGHA1-1 PR8 (no his tag) SEQ ID NO: 137ATGAAATTCTTAGTCAACGTTGCCCTTGTTTTTATGGTCGTATACATTTCTTACATCTATGCGAGCCACAACGGAAAGCTGTGCCGTCTGAAAGGCATAGCGCCACTGCAGCTCGGCAAATGTAACATCGCAGGTTGGCTCCTTGGTAACCCGGAGTGCGACCCCCTCCTCCCTGTACGATCCTGGAGTTATATCGTGGAGACCCCCAATAGCGAGAACGGAATTTGCTACCCAGGAGATTTCATAGACTACGAGGAGTTGCGCGAGCAGCTTTCGTCTGTGAGCAGCTTCGAAAGGTTCGAGATATTCCCGAAGGAGAGCAGCTGGCCGAATCATAACACTAACGGTGTGACAGCCGCCTGCAGTCATGAAGGAAAGAGTTCATTCTATCGCAACCTGCTGTGGTTGACGGAGAAAGAGGGCAGCTACCCTAAGTTGAAGAACTCCTATGTGAACAAAAAAGGCAAGGAGGTTCTGGTGCTGTGGGGCATACACCACCCCCCCAATAGCAAGGAGCAGCAGAATCTGTACCAAAACGAGAATGCCTATGTGAGCGTGGTCACTAGTAACTATAACCGTCGGTTCACTCCCGAGATCGCCGAGCGTCCGAAGGTGAGGGACCAGGCAGGCCGGATGAACTACTACTGGACCCTATTGAAGCCAGGGGACACGATTATCTTCGAGGCAAACGGAAACCTCATAGCGCCGATGTACGCCTTCGCCCTGAGCCGTGGCTTTGGATCGGGGATCATCACGTCTAACGCCTCGATGCACGAATGTAATACCAAATGCCAGACCCCACTGGGTGCTATCAACTCGTCCTTACCCTATCAAAATATACATCCGGTCACCATAGGCGAGTGTCCCAAATATGTCAGATAATAG REV PRIMER SEQ ID NO: 138ACCTGCATGCCTATTATCTGACATATTTGGGACACTCGCCTATGG HA1-1 NC SEQ ID NO: 139ATGAAATTCTTAGTCAACGTTGCCCTTGTTTTTATGGTCGTATACATTTCTTACATCTATGCGAGCCACAATGGTAAGCTGTGTCTGTTGAAGGGAATTGCACCCCTCCAACTGGGCAACTGTTCGGTCGCTGGTTGGATCCTCGGAAACCCAGAATGCGAGCTCCTTATCAGTAAGGAATCTTGGTCTTATATTGTCGAAACCCCGAACCCCGAGAACGGAACATGCTACCCGGGTTACTTTGCTGATTACGAAGAGCTTCGCGAGCAACTCAGCTCCGTATCCTCCTTCGAGCGCTTCGAGATTTTTCCCAAAGAGTCCAGCTGGCCAAATCATACCGTCACCGGCGTGTCGGCCTCCTGTTCCCACAACGGAAAGTCTAGCTTCTATAGAAATCTTCTCTGGCTGACGGGTAAGAATGGTCTTTACCCCAATTTGAGCAAGTCCTACGTCAACAACAAAGAAAAGGAAGTTCTGGTATTGTGGGGTGTGCACCACCCTCCGAACATCGGCAATCAGCGCGCCCTGTATCACACAGAGAACGCGTATGTTTCCGTTGTCTCCTCACATTACTCGAGGCGCTTCACTCCTGAAATAGCTAAGCGTCCGAAAGTGCGTGACCAGGAGGGACGTATCAACTATTATTGGACGCTGTTGGAGCCAGGCGATACAATTATCTTCGAGGCTAACGGTAACCTTATCGCTCCCTGGTACGCCTTCGCCCTGTCGCGTGGTTTCGGTAGTGGAATAATCACTAGTAATGCTCCTATGGACGAGTGTGACGCTAAGTGCCAAACACCTCAGGGCGCTATCAATAGCTCCCTTCCATTCCAGAACGTCCATCCGGTTACCATTGGAGAGTGTCCAAAGTACGTGAGACACCATCATCACCATCACTAA TAGFOR PRIMER SEQ ID NO: 140AGGCAGATCTATGAAATTCTTAGTCAACGTTGCCCTTGTTTTTATGGTCGTATACATTTCTTACATCTATGCGAGCCACAATGGTAAGCTGTGTCTGTTGAAG REV PRIMER SEQ ID NO: 141AGTCGCATGCCTATTAATGGTGGTGATGATGGTGTCTCACGTACTTTGGACACTCTCCAATGG HA0s NCSEQ ID NO: 142AGATCTATGAAGTTTCTCGTGAACGTTGCACTGGTGTTTATGGTAGTCTATATATCGTACATTTATGCTGACACGATTTGCATCGGTTACCATGCGAACAACTCCACGGATACCGTGGACACAGTGTTGGAAAAGAACGTGACCGTCACGCACTCCGTAAACCTTCTGGAGGACAGCCACAATGGTAAGCTGTGTCTGTTGAAGGGAATTGCACCCCTCCAACTGGGCAACTGTTCGGTCGCTGGTTGGATCCTCGGAAACCCAGAATGCGAGCTCCTTATCAGTAAGGAATCTTGGTCTTATATTGTCGAAACCCCGAACCCCGAGAACGGAACATGCTACCCGGGTTACTTTGCTGATTACGAAGAGCTTCGCGAGCAACTCAGCTCCGTATCCTCCTTCGAGCGCTTCGAGATTTTTCCCAAAGAGTCCAGCTGGCCAAATCATACCGTCACCGGCGTGTCGGCCTCCTGTTCCCACAACGGAAAGTCTAGCTTCTATAGAAATCTTCTCTGGCTGACGGGTAAGAATGGTCTTTACCCCAATTTGAGCAAGTCCTACGTCAACAACAAAGAAAAGGAAGTTCTGGTATTGTGGGGTGTGCACCACCCTCCGAACATCGGCAATCAGCGCGCCCTGTATCACACAGAGAACGCGTATGTTTCCGTTGTCTCCTCACATTACTCGAGGCGCTTCACTCCTGAAATAGCTAAGCGTCCGAAAGTGCGTGACCAGGAGGGACGTATCAACTATTATTGGACGCTGTTGGAGCCAGGCGATACAATTATCTTCGAGGCTAACGGTAACCTTATCGCTCCCTGGTACGCCTTCGCCCTGTCGCGTGGTTTCGGTAGTGGAATAATCACTAGTAATGCTCCTATGGACGAGTGTGACGCTAAGTGCCAAACACCTCAGGGCGCTATCAATAGCTCCCTTCCATTCCAGAACGTCCATCCGGTTACCATTGGAGAGTGTCCAAAGTACGTGAGATCGGCCAAACTTCGCATGGTCACGGGTCTGCGCAACATCCCGTCAATCCAATCTAGGGGCCTCTTCGGCGCTATCGCCGGTTTCATTGAGGGCGGTTGGACTGGAATGGTTGACGGATGGTACGGCTATCATCACCAGAACGAACAAGGTTCCGGTTACGCTGCTGACCAGAAATCTACTCAGAACGCGATCAATGGTATCACGAACAAGGTGAACAGCGTCATTGAAAAGATGAATACTCAGTTTACAGCCGTGGGCAAAGAGTTCAATAAACTCGAGAGACGTATGGAAAACCTCAATAAGAAGGTGGATGACGGCTTCCTGGACATTTGGACTTACAACGCCGAGCTGCTGGTCCTGCTCGAGAACGAGAGAACCCTTGACTTCCACGACAGCAACGTCAAGAACCTGTACGAGAAGGTGAAAAGTCAACTTAAAAACAATGCCAAGGAGATTGGTAACGGCTGCTTCGAATTCTACCACAAGTGTAATAATGAGTGCATGGAATCCGTTAAGAACGGCACCTACGATTACCCTAAATACTCAGAGGAGTCCAAGCTTAACCGCGAGAAGATCGACGGCGTAAAACTGGAAAGCATGGGCGTATACCAACACCATCATCACCATCACTAATAGGCATGC HA1-1 VN SEQ ID NO: 143ATGAAATTCTTAGTCAACGTTGCCCTTGTTTTTATGGTCGTATACATTTCTTACATCTATGCGGAGAAGAAACACAACGGTAAGCTTTGCGACTTGGATGGAGTCAAGCCCCTCATACTTAGAGATTGTAGTGTAGCCGGTTGGCTGCTCGGTAACCCAATGTGCGATGAGTTCATCAATGTTCCCGAATGGTCATATATCGTCGAAAAAGCTAATCCTGTCAACGACCTGTGCTACCCCGGTGATTTCAATGACTATGAAGAACTGAAGCACCTGCTCTCCCGCATCAACCATTTCGAGAAAATCCAGATCATTCCCAAGAGTTCCTGGTCTAGCCATGAGGCTAGTCTGGGTGTCTCATCCGCCTGCCCATATCAGGGTAAAAGTTCTTTCTTTAGGAACGTAGTATGGTTGATAAAGAAAAACTCTACATACCCGACCATCAAGCGCTCTTACAACAATACGAACCAAGAGGATCTGCTTGTCCTTTGGGGAATCCATCATCCTAATGATGCTGCCGAACAGACTAAGCTCTACCAAAACCCTACCACTTATATTTCCGTCGGCACCTCTACTCTGAACCAGCGCCTTGTGCCCAGGATCGCTACGAGATCAAAAGTCAACGGCCAATCGGGCCGCATGGAATTCTTCTGGACGATCCTGAAGCCTAATGACGCTATCAACTTCGAGTCAAATGGAAACTTTATCGCTCCCGAGTACGCTTACAAGATCGTCAAGAAGGGCGACTCCACGATTATGAAGTCAGAGTTGGAGTACGGCAACTGCAACACAAAGTGCCAAACTCCTATGGGCGCTATAAATTCTTCAATGCCGTTCCACAACATCCATCCGCTCACGATCGGTGAGTGCCCGAAATATGTAAAGCACCATCACCACCAT CACTAATAGFOR PRIMER SEQ ID NO: 144AGGCAGATCTATGAAATTCTTAGTCAACGTTGCCCTTGTTTTTATGGTCGTATACATTTCTTACATCTATGCGGAGAAGAAACACAACGGTAAGCTTTG REV PRIMER SEQ ID NO: 145AGTCGCATGCCTATTATGGTGGTGATGATGGTGCTTTACATATTTCGGGCACTCACCG HA0s VNSEQ ID NO: 146AGATCTATGAAATTCTTGGTTAATGTAGCCCTGGTGTTTATGGTAGTGTACATTTCATACATTTATGCTGATCAAATCTGCATTGGCTACCATGCCAACAACAGCACCGAGCAAGTTGACACGATCATGGAGAAGAACGTAACCGTCACTCACGCTCAAGACATCCTGGAGAAGAAACACAACGGTAAGCTTTGCGACTTGGATGGAGTCAAGCCCCTCATACTTAGAGATTGTAGTGTAGCCGGTTGGCTGCTCGGTAACCCAATGTGCGATGAGTTCATCAATGTTCCCGAATGGTCATATATCGTCGAAAAAGCTAATCCTGTCAACGACCTGTGCTACCCCGGTGATTTCAATGACTATGAAGAACTGAAGCACCTGCTCTCCCGCATCAACCATTTCGAGAAAATCCAGATCATTCCCAAGAGTTCCTGGTCTAGCCATGAGGCTAGTCTGGGTGTCTCATCCGCCTGCCCATATCAGGGTAAAAGTTCTTTCTTTAGGAACGTAGTATGGTTGATAAAGAAAAACTCTACATACCCGACCATCAAGCGCTCTTACAACAATACGAACCAAGAGGATCTGCTTGTCCTTTGGGGAATCCATCATCCTAATGATGCTGCCGAACAGACTAAGCTCTACCAAAACCCTACCACTTATATTTCCGTCGGCACCTCTACTCTGAACCAGCGCCTTGTGCCCAGGATCGCTACGAGATCAAAAGTCAACGGCCAATCGGGCCGCATGGAATTCTTCTGGACGATCCTGAAGCCTAATGACGCTATCAACTTCGAGTCAAATGGAAACTTTATCGCTCCCGAGTACGCTTACAAGATCGTCAAGAAGGGCGACTCCACGATTATGAAGTCAGAGTTGGAGTACGGCAACTGCAACACAAAGTGCCAAACTCCTATGGGCGCTATAAATTCTTCAATGCCGTTCCACAACATCCATCCGCTCACGATCGGTGAGTGCCCGAAATATGTAAAGTCGAATCGTCTCGTACTGGCGACAGGCCTGAGAAATAGTCCGCAACGTGAACGTCGTCGCAAGAAGAGAGGACTGTTTGGTGCCATTGCAGGCTTTATTGAGGGCGGCTGGCAGGGCATGGTTGACGGATGGTACGGCTACCACCATTCAAACGAGCAGGGATCTGGCTACGCCGCTGACAAAGAAAGCACCCAAAAGGCCATTGATGGAGTGACGAATAAGGTGAATTCGATCATCGACAAAATGAACACGCAATTCGAAGCAGTGGGTCGCGAATTCAATAACCTGGAGCGCCGTATCGAGAATCTGAACAAGAAGATGGAAGACGGCTTTTTGGATGTCTGGACATATAACGCTGAATTGCTGGTCCTCATGGAAAACGAGCGTACCCTTGATTTCCACGACAGCAACGTTAAGAACCTCTACGACAAGGTCAGGCTCCAGCTCAGGGATAACGCCAAGGAATTGGGAAACGGATGCTTCGAGTTCTACCACAAATGCGACAACGAGTGCATGGAGTCAGTCAGGAATGGTACCTACGACTACCCGCAATATTCTGAGGAGGCTCGCTTGAAGCGTGAGGAAATATCGGGTGTTAAATTGGAGAGTATTGGAATCTACCAGCACCATCACCACCATCACTAATAGGCATGC HA1-1 IND SEQ ID NO: 147ATGAAATTCTTAGTCAACGTTGCCCTTGTTTTTATGGTCGTATACATTTCTTACATCTATGCGGAGAAAACCCATAACGGTAAGTTGTGCGACCTTGACGGTGTAAAGCCCCTGATCCTCCGTGACTGCAGTGTTGCTGGTTGGCTTTTGGGCAACCCCATGTGTGACGAATTTATCAACGTGCCTGAATGGTCATACATTGTAGAGAAGGCCAACCCCACGAACGATCTCTGTTATCCCGGCAGCTTCAATGACTATGAGGAACTTAAGCACCTTCTGTCACGTATCAACCACTTCGAAAAGATCCAGATCATCCCGAAGAGCTCCTGGAGCGACCACGAAGCCAGTTCGGGTGTGTCTTCCGCTTGCCCCTACCTCGGTAGCCCTTCCTTCTTCCGTAACGTAGTGTGGCTGATCAAGAAGAATAGCACTTACCCTACAATCAAAAAGTCGTATAACAATACTAACCAAGAGGATCTGCTTGTACTCTGGGGAATTCATCATCCCAACGACGCGGCGGAGCAGACCAGGTTGTACCAGAACCCCACCACTTACATCTCCATCGGTACGTCCACACTGAATCAGCGTCTGGTCCCCAAGATCGCAACCAGGTCCAAGGTTAACGGTCAGTCCGGTCGTATGGAGTTCTTCTGGACCATCCTGAAGCCCAACGACGCCATCAACTTCGAGTCCAACGGTAATTTCATTGCTCCGGAGTACGCCTACAAGATAGTTAAGAAGGGTGATTCAGCGATCATGAAGTCGGAACTTGAGTATGGCAACTGCAACACTAAATGCCAAACTCCAATGGGCGCTATCAACTCCAGTATGCCATTCCATAACATCCACCCATTGACAATCGGTGAATGTCCCAAGTACGTGAAGCACCACCATCACCAT CACTAATAGFOR PRIMER SEQ ID NO: 148AGGCAGATCTATGAAATTCTTAGTCAACGTTGCCCTTGTTTTTATGGTCGTATACATTTCTTACATCTATGCGGAGAAAACCCATAACGGTAAGTTGTG REV PRIMER SEQ ID NO: 149AGTCGCATGCCTATTAATGGTGGTGATGATGGTGCTTCACGTACTTGGGACATTCACCGA TTGHA0s IND SEQ ID NO: 150AGATCTATGAAGTTCCTGGTCAATGTAGCCTTGGTATTTATGGTAGTCTATATCTCGTACATTTACGCAGACCAGATTTGTATTGGATATCACGCTAACAACAGCACAGAGCAGGTAGATACTATTATGGAGAAAAATGTTACCGTCACTCACGCCCAGGACATCCTGGAGAAAACCCATAACGGTAAGTTGTGCGACCTTGACGGTGTAAAGCCCCTGATCCTCCGTGACTGCAGTGTTGCTGGTTGGCTTTTGGGCAACCCCATGTGTGACGAATTTATCAACGTGCCTGAATGGTCATACATTGTAGAGAAGGCCAACCCCACGAACGATCTCTGTTATCCCGGCAGCTTCAATGACTATGAGGAACTTAAGCACCTTCTGTCACGTATCAACCACTTCGAAAAGATCCAGATCATCCCGAAGAGCTCCTGGAGCGACCACGAAGCCAGTTCGGGTGTGTCTTCCGCTTGCCCCTACCTCGGTAGCCCTTCCTTCTTCCGTAACGTAGTGTGGCTGATCAAGAAGAATAGCACTTACCCTACAATCAAAAAGTCGTATAACAATACTAACCAAGAGGATCTGCTTGTACTCTGGGGAATTCATCATCCCAACGACGCGGCGGAGCAGACCAGGTTGTACCAGAACCCCACCACTTACATCTCCATCGGTACGTCCACACTGAATCAGCGTCTGGTCCCCAAGATCGCAACCAGGTCCAAGGTTAACGGTCAGTCCGGTCGTATGGAGTTCTTCTGGACCATCCTGAAGCCCAACGACGCCATCAACTTCGAGTCCAACGGTAATTTCATTGCTCCGGAGTACGCCTACAAGATAGTTAAGAAGGGTGATTCAGCGATCATGAAGTCGGAACTTGAGTATGGCAACTGCAACACTAAATGCCAAACTCCAATGGGCGCTATCAACTCCAGTATGCCATTCCATAACATCCACCCATTGACAATCGGTGAATGTCCCAAGTACGTGAAGAGCAACAGGTTGGTATTGGCCACCGGTTTGAGAAACAGCCCCCAGAGAGAGTCGCGTCGTAAAAAGCGCGGCTTGTTCGGAGCCATCGCTGGCTTCATAGAGGGTGGTTGGCAGGGAATGGTCGATGGTTGGTATGGTTATCATCATTCCAACGAGCAGGGAAGTGGTTACGCCGCCGACAAAGAATCGACCCAGAAGGCTATTGACGGCGTCACAAACAAAGTAAACTCTATCATTGATAAGATGAACACCCAGTTCGAGGCTGTAGGTAGAGAATTCAACAACCTCGAAAGACGTATTGAGAACCTGAACAAGAAAATGGAGGATGGCTTCCTGGACGTGTGGACCTACAATGCTGAGCTGTTGGTCCTTATGGAGAACGAGCGTACCCTCGATTTCCATGACTCAAACGTGAAGAACCTGTATGACAAGGTGCGTTTGCAACTGAGGGACAACGCAAAGGAGCTTGGAAACGGTTGTTTCGAATTTTATCATAAGTGCGACAATGAGTGTATGGAGTCGATTAGAAATGGCACGTACAACTACCCTCAATACAGCGAAGAAGCTCGTCTCAAACGTGAGGAAATCAGCGGCGTCAAGCTCGAATCAATCGGTACCTATCAGCACCACCATCACCATCACTAATAGGCATGC STF2.HA1-1His(PR8) SEQ ID NO: 151MKFLVNVALVFMVVYISYIYAAQVINTNSLSLLTQNNLNKSQSALGTAIERLSSGLRINSAKDDAAGQAIANRFTANIKGLTQASRNANDGISIAQTTEGALNEINNNLQRVRELAVQSANSTNSQSDLDSIQAEITQRLNEIDRVSGQTQFNGVKVLAQDNTLTIQVGANDGETIDIDLKQINSQTLGLDSLNVQKAYDVKDTAVTTKAYANNGTTLDVSGLDDAAIKAATGGTNGTASVTGGAVKFDADNNKYFVTIGGFTGADAAKNGDYEVNVATDGTVTLAAGATKTTMPAGATTKTEVQELKDTPAVVSADAKNALIAGGVDATDANGAELVKMSYTDKNGKTIEGGYALKAGDKYYAADYDEATGAIKAKTTSYTAADGTTKTAANQLGGVDGKTEVVTIDGKTYNASKAAGHDFKAQPELAEAAAKTTENPLQKIDAALAQVDALRSDLGAVQNRFNSAITNLGNTVNNLSEARSRIEDSDYATEVSNMSRAQILQQAGTSVLAQANQVPQNVLSLLASGSGSGSSHNGKLCRLKGIAPLQLGKCNIAGWLLGNPECDPLLPVRSWSYIVETPNSENGICYPGDFIDYEELREQLSSVSSFERFEIFPKESSWPNHNTNGVTAACSHEGKSSFYRNLLWLTEKEGSYPKLKNSYVNKKGKEVLVLWGIHHPPNSKEQQNLYQNENAYVSVVTSNYNRRFTPEIAERPKVRDQAGRMNYYWTLLKPGDTIIFEANGNLIAPMYAFALSRGFGSGIITSNASMHECNTKCQTPLGAINSSLPYQNIHPVTIGECPKYVRHHHHHH ngSTF2.HA1-1His(PR8)SEQ ID NO: 152MKFLVNVALVFMVVYISYIYAAQVINTNSLSLLTQNNLQKSQSALGTAIERLSSGLRINSAKDDAAGQAIANRFTANIKGLTQASRNANDGISIAQTTEGALNEINNNLQRVRELAVQSAQSTNSQSDLDSIQAEITQRLNEIDRVSGQTQFNGVKVLAQDNTLTIQVGANDGETIDIDLKQINSQTLGLDSLNVQKAYDVKDTAVTTKAYANQGTTLDVSGLDDAAIKAATGGTQGTASVTGGAVKFDADNNKYFVTIGGFTGADAAKNGDYEVNVATDGTVTLAAGATKTTMPAGATTKTEVQELKDTPAVVSADAKNALIAGGVDATDANGAELVKMSYTDKNGKTIEGGYALKAGDKYYAADYDEATGAIKAKTTSYTAADGTTKTAANQLGGVDGKTEVVTIDGKTYQASKAAGHDFKAQPELAEAAAKTTENPLQKIDAALAQVDALRSDLGAVQNRFNSAITNLGNTVNQLSEARSRIEDSDYATEVSQMSRAQILQQAGTSVLAQANQVPQNVLSLLASGSGSGSSHNGKLCRLKGIAPLQLGKCNIAGWLLGNPECDPLLPVRSWSYIVETPNSENGICYPGDFIDYEELREQLSSVSSFERFEIFPKESSWPNHNTNGVTAACSHEGKSSFYRNLLWLTEKEGSYPKLKNSYVNKKGKEVLVLWGIHHPPNSKEQQNLYQNENAYVSVVTSNYNRRFTPEIAERPKVRDQAGRMNYYWTLLKPGDTIIFEANGNLIAPMYAFALSRGFGSGIITSQASMHECNTKCQTPLGAIQSSLPYQNIHPVTIGECPKYVRHHHHHH STF2.HA1-2His(PR8)SEQ ID NO: 153MKFLVNVALVFMVVYISYIYAAQVINTNSLSLLTQNNLNKSQSALGTAIERLSSGLRINSAKDDAAGQAIANRFTANIKGLTQASRNANDGISIAQTTEGALNEINNNLQRVRELAVQSANSTNSQSDLDSIQAEITQRLNEIDRVSGQTQFNGVKVLAQDNTLTIQVGANDGETIDIDLKQINSQTLGLDSLNVQKAYDVKDTAVTTKAYANNGTTLDVSGLDDAAIKAATGGTNGTASVTGGAVKFDADNNKYFVTIGGFTGADAAKNGDYEVNVATDGTVTLAAGATKTTMPAGATTKTEVQELKDTPAVVSADAKNALIAGGVDATDANGAELVKMSYTDKNGKTIEGGYALKAGDKYYAADYDEATGAIKAKTTSYTAADGTTKTAANQLGGVDGKTEVVTIDGKTYNASKAAGHDFKAQPELAEAAAKTTENPLQKIDAALAQVDALRSDLGAVQNRFNSAITNLGNTVNNLSEARSRIEDSDYATEVSNMSRAQILQQAGTSVLAQANQVPQNVLSLLASGSGSGSKGIAPQLGKCNIAGWLLGNPECDPLLPVRSWSYIVETPNSENGICYPGDFIDYEELREQLSSVSSFERFEIFPKESSWPNHNTNGVTAACSHEGKSSFYRNLLWLTEKEGSYPKLKNSYVNKKGKEVLVLWGIHHPPNSKEQQNLYQNENAYVSVVTSNYNRRFTPEIAERPKVRDQAGRMNYYWTLLKPGDTIIFEANGNLIAPMYAFALSRGFGSGIITSHHHHHHngSTF2.HA1-2His(PR8) SEQ ID NO: 154MKFLVNVALVFMVVYISYIYAAQVINTNSLSLLTQNNLQKSQSALGTAIERLSSGLRINSAKDDAAGQAIANRFTANIKGLTQASRNANDGISIAQTTEGALNEINNNLQRVRELAVQSAQSTNSQSDLDSIQAEITQRLNEIDRVSGQTQFNGVKVLAQDNTLTIQVGANDGETIDIDLKQINSQTLGLDSLNVQKAYDVKDTAVTTKAYANQGTTLDVSGLDDAAIKAATGGTQGTASVTGGAVKFDADNNKYFVTIGGFTGADAAKNGDYEVNVATDGTVTLAAGATKTTMPAGATTKTEVQELKDTPAVVSADAKNALIAGGVDATDANGAELVKMSYTDKNGKTIEGGYALKAGDKYYAADYDEATGAIKAKTTSYTAADGTTKTAANQLGGVDGKTEVVTIDGKTYQASKAAGHDFKAQPELAEAAAKTTENPLQKIDAALAQVDALRSDLGAVQNRFNSAITNLGNTVNQLSEARSRIEDSDYATEVSQMSRAQILQQAGTSVLAQANQVPQNVLSLLASGSGSGSKGIAPLQLGKCNIAGWLLGNPECDPLLPVRSWSYIVETPNSENGICYPGDFIDYEELREQLSSVSSFERFEIFPKESSWPNHNTNGVTAACSHEGKSSFYRNLLWLTEKEGSYPKLKNSYVNKKGKEVLVLWGIHHPPNSKEQQNLYQNENAYVSVVTSNYNRRFTPEIAERPKVRDQAGRMNYYWTLLKPGDTIIFEANGNLIAPMYAFALSRGFGSGIITSHHHHHHSTF2.HA1-2mutHis(PR8) SEQ ID NO: 155MKFLVNVALVFMVVYISYIYAAQVINTNSLSLLTQNNLNKSQSALGTAIERLSSGLRINSAKDDAAGQAIANRFTANIKGLTQASRNANDGISIAQTTEGALNEINNNLQRVRELAVQSANSTNSQSDLDSIQAEITQRLNEIDRVSGQTQFNGVKVLAQDNTLTIQVGANDGETIDIDLKQINSQTLGLDSLNVQKAYDVKDTAVTTKAYANNGTTLDVSGLDDAAIKAATGGTNGTASVTGGAVKFDADNNKYFVTIGGFTGADAAKNGDYEVNVATDGTVTLAAGATKTTMPAGATTKTEVQELKDTPAVVSADAKNALIAGGVDATDANGAELVKMSYTDKNGKTIEGGYALKAGDKYYAADYDEATGAIKAKTTSYTAADGTTKTAANQLGGVDGKTEVVTIDGKTYNASKAAGHDFKAQPELAEAAAKTTENPLQKIDAALAQVDALRSDLGAVQNRFNSAITNLGNTVNNLSEARSRIEDSDYATEVSNMSRAQILQQAGTSVLAQANQVPQNVLSLLASGSGSGSKGAAPLQLGKCNIAGWLLGNPECDPLLPVRSWSDIAETPNSENGICYPGDFIDYEELREQLSSVSSFERFEIFPKESSWPNHNTNGVTAACSHEGKSSFYRNLLWLTEKEGSYPKLKNSYVNKKGKEVLVLWGIHHPPNSKEQQNLYQNENAYVSVVTSNYNRRFTPEIAERPKVRDQAGRMNYYWTLLKPGDTIIFEANGNLIAPMYAFALSRGFGSGIITSHHHHHHngSTF2.HA1-2mutHis(PR8) SEQ ID NO: 156MKFLVNVALVFMVVYISYIYAAQVINTNSLSLLTQNNLQKSQSALGTAIERLSSGLRINSAKDDAAGQAIANRFTANIKGLTQASRNANDGISIAQTTEGALNEINNNLQRVRELAVQSAQSTNSQSDLDSIQAEITQRLNEIDRVSGQTQFNGVKVLAQDNTLTIQVGANDGETIDIDLKQINSQTLGLDSLNVQKAYDVKDTAVTTKAYANQGTTLDVSGLDDAAIKAATGGTQGTASVTGGAVKFDADNNKYFVTIGGFTGADAAKNGDYEVNVATDGTVTLAAGATKTTMPAGATTKTEVQELKDTPAVVSADAKNALIAGGVDATDANGAELVKMSYTDKNGKTIEGGYALKAGDKYYAADYDEATGAIKAKTTSYTAADGTTKTAANQLGGVDGKTEVVTIDGKTYQASKAAGHDFKAQPELAEAAAKTTENPLQKIDAALAQVDALRSDLGAVQNRFNSAITNLGNTVNQLSEARSRIEDSDYATEVSQMSRAQILQQAGTSVLAQANQVPQNVLSLLASGSGSGSKGAAPQLGKCNIAGWLLGNPECDPLLPVRSWSDIAETPNSENGICYPGDFIDYEELREQLSSVSSFERFEIFPKESSWPNHNTNGVTAACSHEGKSSFYRNLLWLTEKEGSYPKLKNSYVNKKGKEVLVLWGIHHPPNSKEQQNLYQNENAYVSVVTSNYNRRFTPEIAERPKVRDQAGRMNYYWTLLKPGDTIIFEANGNLIAPMYAFALSRGFGSGIITSHHHHHHSTF2.HA1-3His(PR8) SEQ ID NO: 157MKFLVNVALVFMVVYISYIYAAQVINTNSLSLLTQNNLNKSQSALGTAIERLSSGLRINSAKDDAAGQAIANRFTANIKGLTQASRNANDGISIAQTTEGALNEINNNLQRVRELAVQSANSTNSQSDLDSIQAEITQRLNEIDRVSGQTQFNGVKVLAQDNTLTIQVGANDGETIDIDLKQINSQTLGLDSLNVQKAYDVKDTAVTTKAYANNGTTLDVSGLDDAAIKAATGGTNGTASVTGGAVKFDADNNKYFVTIGGFTGADAAKNGDYEVNVATDGTVTLAAGATKTTMPAGATTKTEVQELKDTPAVVSADAKNALIAGGVDATDANGAELVKMSYTDKNGKTIEGGYALKAGDKYYAADYDEATGAIKAKTTSYTAADGTTKTAANQLGGVDGKTEVVTIDGKTYNASKAAGHDFKAQPELAEAAAKTTENPLQKIDAALAQVDALRSDLGAVQNRFNSAITNLGNTVNNLSEARSRIEDSDYATEVSNMSRAQILQQAGTSVLAQANQVPQNVLSLLASGSGSGSNSENGCYPGDFIDYEELREQLSSVSSFERFEIFPKESSWPNHNTNGVTAACSHEGKSSFYRNLLWLTEKEGSYPKLKNSYVNKKGKEVLVLWGIHHPPNSKEQQNLYQNENAYVSVVTSNYNRRFTPEIAERPKVRDQAGRMNYYWTLLKPGDTIIFEANGNLIAPMYAFALSRGHHHHHH STF2.HA1-3mutHis(PR8) SEQ ID NO: 158MKFLVNVALVFMVVYISYIYAAQVINTNSLSLLTQNNLNKSQSALGTAIERLSSGLRINSAKDDAAGQAIANRFTANIKGLTQASRNANDGISIAQTTEGALNEINNNLQRVRELAVQSANSTNSQSDLDSIQAEITQRLNEIDRVSGQTQFNGVKVLAQDNTLTIQVGANDGETIDIDLKQINSQTLGLDSLNVQKAYDVKDTAVTTKAYANNGTTLDVSGLDDAAIKAATGGTNGTASVTGGAVKFDADNNKYFVTIGGFTGADAAKNGDYEVNVATDGTVTLAAGATKTTMPAGATTKTEVQELKDTPAVVSADAKNALIAGGVDATDANGAELVKMSYTDKNGKTIEGGYALKAGDKYYAADYDEATGAIKAKTTSYTAADGTTKTAANQLGGVDGKTEVVTIDGKTYNASKAAGHDFKAQPELAEAAAKTTENPLQKIDAALAQVDALRSDLGAVQNRFNSAITNLGNTVNNLSEARSRIEDSDYATEVSNMSRAQILQQAGTSVLAQANQVPQNVLSLLASGSGSGSNSENEICYPGDFIDKEELREQLSSVSSFERFEIFPKESSWPNHNTNGVTAACSHEGKSSFYRNLLWLTEKEGSYPKLKNSYVNKKGKEVLVLWGIHHPPNSKEQQNLYQNENAYVSVVTSNYNRRFTPEIAERPKVRDQAGRMNYYWTLLKPGDTIIFEANGNLIAPMYAAALSRGHHHHHH STF2.HA1-2(IND) SEQ ID NO: 159MAQVINTNSLSLLTQNNLNKSQSALGTAIERLSSGLRINSAKDDAAGQAIANRFTANIKGLTQASRNANDGISIAQTTEGALNEINNNLQRVRELAVQSANSTNSQSDLDSIQAEITQRLNEIDRVSGQTQFNGVKVLAQDNTLTIQVGANDGETIDIDLKQINSQTLGLDSLNVQKAYDVKDTAVTTKAYANNGTTLDVSGLDDAAIKAATGGTNGTASVTGGAVKFDADNNKYFVTIGGFTGADAAKNGDYEVNVATDGTVTLAAGATKTTMPAGATTKTEVQELKDTPAVVSADAKNALIAGGVDATDANGAELVKMSYTDKNGKTIEGGYALKAGDKYYAADYDEATGAIKAKTTSYTAADGTTKTAANQLGGVDGKTEVVTIDGKTYNASKAAGHDFKAQPELAEAAAKTTENPLQKIDAALAQVDALRSDLGAVQNRFNSAITNLGNTVNNLSEARSRIEDSDYATEVSNMSRAQILQQAGTSVLAQANQVPQNVLSLLAGVKPLILRDCSVAGWLLGNPMCDEFINVPEWSYIVEKANPTNDLCYPGSFNDYEELKHLLSRINHFEKIQIIPKSSWSDHEASSGVSSACPYLGSPSFFRNVVWLIKKNSTYPTIKKSYNNTNQEDLLVLWGIHHPNDAAEQTRLYQNPTTYISIGTSTLNQRLVPKIATRSKVNGQSGRMEFFWTILKPNDAINFESNGNFIAPEYAYKIVKKGDSAIMKSE Drosophila STF2Δ.HA0shis SEQ ID NO: 160ATGGCACAAGTAATCAACACTAACAGTCTGTCGCTGCTGACCCAGAATAACCTGAACAAATCCCAGTCCGCACTGGGCACCGCTATCGAGCGTCTGTCTTCTGGTCTGCGTATCAACAGCGCGAAAGACGATGCGGCAGGTCAGGCGATTGCTAACCGTTTCACCGCGAACATCAAAGGTCTGACTCAGGCTTCCCGTAACGCTAACGACGGTATCTCCATTGCGCAGACCACTGAAGGCGCGCTGAACGAAATCAACAACAACCTGCAGCGTGTGCGTGAACTGGCGGTTCAGTCTGCTAACAGCACCAACTCCCAGTCTGACCTCGACTCCATCCAGGCTGAAATCACCCAGCGCCTGAACGAAATCGACCGTGTATCCGGCCAGACTCAGTTCAACGGCGTGAAAGTCCTGGCGCAGGACAACACCCTGACCATCCAGGTTGGCGCCAACGACGGTGAAACTATCGATATCGATCTGAAGCAGATCAACTCTCAGACCCTGGGTCTGGACTCACTGAACGTGCATGGAGCGCCGGTGGATCCTGCTAGCCCATGGACCGAAAACCCGCTGCAGAAAATTGATGCCGCGCTGGCGCAGGTGGATGCGCTGCGCTCTGATCTGGGTGCGGTACAAAACCGTTTCAACTCTGCTATCACCAACCTGGGCAATACCGTAAACAATCTGTCTGAAGCGCGTAGCCGTATCGAAGATTCCGACTACGCGACCGAAGTTTCCAACATGTCTCGCGCGCAGATTTTGCAGCAGGCCGGTACTTCCGTTCTGGCGCAGGCTAACCAGGTCCCGCAGAACGTGCTGTCTCTGTTACGTGAATTCTCTAGATATCCAGCACAGTGGCGGCCGCTCGACACAATATGTATAGGCTACCATGCGAACAATTCAACCGACACTGTTGACACAGTACTCGAGAAGAATGTGACAGTGACACACTCTGTTAACCTGCTCGAAGACAGCCACAACGGAAAACTATGTAGATTAAAAGGAATAGCCCCACTACAATTGGGGAAATGTAACATCGCCGGATGGCTTTTGGGAAACCCAGAATGCGACCCACTGCTTCCAGTGAGATCATGGTCCTACATTGTAGAAACACCAAACTCTGAGAATGGAATATGTTATCCAGGAGATTTCATCGACTATGAGGAGCTGAGGGAGCAATTGAGCTCAGTGTCATCATTCGAAAGATTCGAAATATTTCCCAAAGAAAGCTCATGGCCCAACCACAACACAAACGGAGTAACGGCAGCATGCTCCCATGAGGGGAAAAGCAGTTTTTACAGAAATTTGCTATGGCTGACGGAGAAGGAGGGCTCATACCCAAAGCTGAAAAATTCTTATGTGAACAAAAAAGGGAAAGAAGTCCTTGTACTGTGGGGTATTCATCACCCGCCTAACAGTAAGGAACAACAGAATCTCTATCAGAATGAAAATGCTTATGTCTCTGTAGTGACTTCAAATTATAACAGGAGATTTACCCCGGAAATAGCAGAAAGACCCAAAGTAAGAGATCAAGCTGGGAGGATGAACTATTACTGGACCTTGCTAAAACCCGGAGACACAATAATATTTGAGGCAAATGGAAATCTAATAGCACCAATGTATGCTTTCGCACTGAGTAGAGGCTTTGGGTCCGGCATCATCACCTCAAACGCATCAATGCATGAGTGTAACACGAAGTGTCAAACACCCCTGGGAGCTATAAACAGCAGTCTCCCTTACCAGAATATACACCCAGTCACAATAGGAGAGTGCCCAAAATACGTCAGGAGTGCCAAATTGAGGATGGTTACAGGACTAAGGAACATTCCGTCCATTCAATCCAGAGGTCTATTTGGAGCCATTGCCGGTTTTATTGAAGGGGGATGGACTGGAATGATAGATGGATGGTATGGTTATCATCATCAGAATGAACAGGGATCAGGCTATGCAGCGGATCAAAAAAGCACACAAAATGCCATTAACGGGATTACAAACAAGGTGAACACTGTTATCGAGAAAATGAACATTCAATTCACAGCTGTGGGTAAAGAATTCAACAAATTAGAAAAAAGGATGGAAAATTTAAATAAAAAAGTTGATGATGGATTTCTGGACATTTGGACATATAATGCAGAATTGTTAGTTCTACTGGAAAATGAAAGGACTCTGGATTTCCATGACTCAAATGTGAAGAATCTGTATGAGAAAGTAAAAAGCCAATTAAAGAATAATGCCAAAGAAATCGGAAATGGATGTTTTGAGTTCTACCACAAGTGTGACAATGAATGCATGGAAAGTGTAAGAAATGGGACTTATGATTATCCCAAATATTCAGAAGAGTCAAAGTTGAACAGGGAAAAGGTAGATGGAGTGAAATTGGAATCAATGGGGATCTATCAGACGCGTACCGGTCATCATCACCATCACCATTGA FOR PRIMER SEQ ID NO: 161ATTCTCCGGCGGCCGCTCGACACAATATGTATAGGCTACC REV PRIMER SEQ ID NO: 162AGTCTTGCGGCCGCCTATTAATGGTGATGGTGATGATGCTGATAGATCCCCATTGATTCCHA0s PR8 template SEQ ID NO: 163GACACAATATGTATAGGCTACCATGCGAACAATTCAACCGACACTGTTGACACAGTACTCGAGAAGAATGTGACAGTGACACACTCTGTTAACCTGCTCGAAGACAGCCACAACGGAAAACTATGTAGATTAAAAGGAATAGCCCCACTACAATTGGGGAAATGTAACATCGCCGGATGGCTTTTGGGAAACCCAGAATGCGACCCACTGCTTCCAGTGAGATCATGGTCCTACATTGTAGAAACACCAAACTCTGAGAATGGAATATGTTATCCAGGAGATTTCATCGACTATGAGGAGCTGAGGGAGCAATTGAGCTCAGTGTCATCATTCGAAAGATTCGAAATATTTCCCAAAGAAAGCTCATGGCCCAACCACAACACAAACGGAGTAACGGCAGCATGCTCCCATGAGGGGAAAAGCAGTTTTTACAGAAATTTGCTATGGCTGACGGAGAAGGAGGGCTCATACCCAAAGCTGAAAAATTCTTATGTGAACAAAAAAGGGAAAGAAGTCCTTGTACTGTGGGGTATTCATCACCCGCCTAACAGTAAGGAACAACAGAATCTCTATCAGAATGAAAATGCTTATGTCTCTGTAGTGACTTCAAATTATAACAGGAGATTTACCCCGGAAATAGCAGAAAGACCCAAAGTAAGAGATCAAGCTGGGAGGATGAACTATTACTGGACCTTGCTAAAACCCGGAGACACAATAATATTTGAGGCAAATGGAAATCTAATAGCACCAATGTATGCTTTCGCACTGAGTAGAGGCTTTGGGTCCGGCATCATCACCTCAAACGCATCAATGCATGAGTGTAACACGAAGTGTCAAACACCCCTGGGAGCTATAAACAGCAGTCTCCCTTACCAGAATATACACCCAGTCACAATAGGAGAGTGCCCAAAATACGTCAGGAGTGCCAAATTGAGGATGGTTACAGGACTAAGGAACATTCCGTCCATTCAATCCAGAGGTCTATTTGGAGCCATTGCCGGTTTTATTGAAGGGGGATGGACTGGAATGATAGATGGATGGTATGGTTATCATCATCAGAATGAACAGGGATCAGGCTATGCAGCGGATCAAAAAAGCACACAAAATGCCATTAACGGGATTACAAACAAGGTGAACACTGTTATCGAGAAAATGAACATTCAATTCACAGCTGTGGGTAAAGAATTCAACAAATTAGAAAAAAGGATGGAAAATTTAAATAAAAAAGTTGATGATGGATTTCTGGACATTTGGACATATAATGCAGAATTGTTAGTTCTACTGGAAAATGAAAGGACTCTGGATTTCCATGACTCAAATGTGAAGAATCTGTATGAGAAAGTAAAAAGCCAATTAAAGAATAATGCCAAAGAAATCGGAAATGGATGTTTTGAGTTCTACCACAAGTGTGACAATGAATGCATGGAAAGTGTAAGAAATGGGACTTATGATTATCCCAAATATTCAGAAGAGTCAAAGTTGAACAGGGAAAAGGTAGATGGAGTGAAATTGGAATCAATGGGGATCTATCAG STF2Δ.HA0s SEQ ID NO: 164ATGGCACAAGTAATCAACACTAACAGTCTGTCGCTGCTGACCCAGAATAACCTGAACAAATCCCAGTCCGCACTGGGCACCGCTATCGAGCGTCTGTCTTCTGGTCTGCGTATCAACAGCGCGAAAGACGATGCGGCAGGTCAGGCGATTGCTAACCGTTTCACCGCGAACATCAAAGGTCTGACTCAGGCTTCCCGTAACGCTAACGACGGTATCTCCATTGCGCAGACCACTGAAGGCGCGCTGAACGAAATCAACAACAACCTGCAGCGTGTGCGTGAACTGGCGGTTCAGTCTGCTAACAGCACCAACTCCCAGTCTGACCTCGACTCCATCCAGGCTGAAATCACCCAGCGCCTGAACGAAATCGACCGTGTATCCGGCCAGACTCAGTTCAACGGCGTGAAAGTCCTGGCGCAGGACAACACCCTGACCATCCAGGTTGGCGCCAACGACGGTGAAACTATCGATATCGATCTGAAGCAGATCAACTCTCAGACCCTGGGTCTGGACTCACTGAACGTGCATGGAGCGCCGGTGGATCCTGCTAGCCCATGGACCGAAAACCCGCTGCAGAAAATTGATGCCGCGCTGGCGCAGGTGGATGCGCTGCGCTCTGATCTGGGTGCGGTACAAAACCGTTTCAACTCTGCTATCACCAACCTGGGCAATACCGTAAACAATCTGTCTGAAGCGCGTAGCCGTATCGAAGATTCCGACTACGCGACCGAAGTTTCCAACATGTCTCGCGCGCAGATTTTGCAGCAGGCCGGTACTTCCGTTCTGGCGCAGGCTAACCAGGTCCCGCAGAACGTGCTGTCTCTGTTACGTGAATTCTCTAGATATCCAGCACAGTGGCGGCCGCTCGACACAATATGTATAGGCTACCATGCGAACAATTCAACCGACACTGTTGACACAGTACTCGAGAAGAATGTGACAGTGACACACTCTGTTAACCTGCTCGAAGACAGCCACAACGGAAAACTATGTAGATTAAAAGGAATAGCCCCACTACAATTGGGGAAATGTAACATCGCCGGATGGCTTTTGGGAAACCCAGAATGCGACCCACTGCTTCCAGTGAGATCATGGTCCTACATTGTAGAAACACCAAACTCTGAGAATGGAATATGTTATCCAGGAGATTTCATCGACTATGAGGAGCTGAGGGAGCAATTGAGCTCAGTGTCATCATTCGAAAGATTCGAAATATTTCCCAAAGAAAGCTCATGGCCCAACCACAACACAAACGGAGTAACGGCAGCATGCTCCCATGAGGGGAAAAGCAGTTTTTACAGAAATTTGCTATGGCTGACGGAGAAGGAGGGCTCATACCCAAAGCTGAAAAATTCTTATGTGAACAAAAAAGGGAAAGAAGTCCTTGTACTGTGGGGTATTCATCACCCGCCTAACAGTAAGGAACAACAGAATCTCTATCAGAATGAAAATGCTTATGTCTCTGTAGTGACTTCAAATTATAACAGGAGATTTACCCCGGAAATAGCAGAAAGACCCAAAGTAAGAGATCAAGCTGGGAGGATGAACTATTACTGGACCTTGCTAAAACCCGGAGACACAATAATATTTGAGGCAAATGGAAATCTAATAGCACCAATGTATGCTTTCGCACTGAGTAGAGGCTTTGGGTCCGGCATCATCACCTCAAACGCATCAATGCATGAGTGTAACACGAAGTGTCAAACACCCCTGGGAGCTATAAACAGCAGTCTCCCTTACCAGAATATACACCCAGTCACAATAGGAGAGTGCCCAAAATACGTCAGGAGTGCCAAATTGAGGATGGTTACAGGACTAAGGAACATTCCGTCCATTCAATCCAGAGGTCTATTTGGAGCCATTGCCGGTTTTATTGAAGGGGGATGGACTGGAATGATAGATGGATGGTATGGTTATCATCATCAGAATGAACAGGGATCAGGCTATGCAGCGGATCAAAAAAGCACACAAAATGCCATTAACGGGATTACAAACAAGGTGAACACTGTTATCGAGAAAATGAACATTCAATTCACAGCTGTGGGTAAAGAATTCAACAAATTAGAAAAAAGGATGGAAAATTTAAATAAAAAAGTTGATGATGGATTTCTGGACATTTGGACATATAATGCAGAATTGTTAGTTCTACTGGAAAATGAAAGGACTCTGGATTTCCATGACTCAAATGTGAAGAATCTGTATGAGAAAGTAAAAAGCCAATTAAAGAATAATGCCAAAGAAATCGGAAATGGATGTTTTGAGTTCTACCACAAGTGTGACAATGAATGCATGGAAAGTGTAAGAAATGGGACTTATGATTATCCCAAATATTCAGAAGAGTCAAAGTTGAACAGGGAAAAGGTAGATGGAGTGAAATTGGAATCAATGGGGATCTATCAG TAGFOR PRIMER SEQ ID NO: 165 ATTCTCCGGCGGCCGCTCGACACAATATGTATAGGCTACCREV PRIMER SEQ ID NO: 166AGTCTTGCGGCCGCCTATTAATGGTGATGGTGATGATGCTGATAGATCCCCATTGATTCC HA0s.STF2ΔSEQ ID NO: 167GACACAATATGTATAGGCTACCATGCGAACAATTCAACCGACACTGTTGACACAGTACTCGAGAAGAATGTGACAGTGACACACTCTGTTAACCTGCTCGAAGACAGCCACAACGGAAAACTATGTAGATTAAAAGGAATAGCCCCACTACAATTGGGGAAATGTAACATCGCCGGATGGCTTTTGGGAAACCCAGAATGCGACCCACTGCTTCCAGTGAGATCATGGTCCTACATTGTAGAAACACCAAACTCTGAGAATGGAATATGTTATCCAGGAGATTTCATCGACTATGAGGAGCTGAGGGAGCAATTGAGCTCAGTGTCATCATTCGAAAGATTCGAAATATTTCCCAAAGAAAGCTCATGGCCCAACCACAACACAAACGGAGTAACGGCAGCATGCTCCCATGAGGGGAAAAGCAGTTTTTACAGAAATTTGCTATGGCTGACGGAGAAGGAGGGCTCATACCCAAAGCTGAAAAATTCTTATGTGAACAAAAAAGGGAAAGAAGTCCTTGTACTGTGGGGTATTCATCACCCGCCTAACAGTAAGGAACAACAGAATCTCTATCAGAATGAAAATGCTTATGTCTCTGTAGTGACTTCAAATTATAACAGGAGATTTACCCCGGAAATAGCAGAAAGACCCAAAGTAAGAGATCAAGCTGGGAGGATGAACTATTACTGGACCTTGCTAAAACCCGGAGACACAATAATATTTGAGGCAAATGGAAATCTAATAGCACCAATGTATGCTTTCGCACTGAGTAGAGGCTTTGGGTCCGGCATCATCACCTCAAACGCATCAATGCATGAGTGTAACACGAAGTGTCAAACACCCCTGGGAGCTATAAACAGCAGTCTCCCTTACCAGAATATACACCCAGTCACAATAGGAGAGTGCCCAAAATACGTCAGGAGTGCCAAGTTGAGGATGGTTACAGGACTAAGGAACATTCCGTCCATTCAATCCAGAGGTCTATTTGGAGCCATTGCCGGTTTTATTGAAGGGGGATGGACTGGAATGATAGATGGATGGTATGGTTATCATCATCAGAATGAACAGGGATCAGGCTATGCAGCGGATCAAAAAAGCACACAAAATGCCATTAACGGGATTACAAACAAGGTGAACACTGTTATCGAGAAAATGAACATTCAATTCACAGCTGTGGGTAAAGAATTCAACAAATTAGAAAAAAGGATGGAAAATTTAAATAAAAAAGTTGATGATGGATTTCTGGACATTTGGACATATAATGCAGAATTGTTAGTTCTACTGGAAAATGAAAGGACTCTGGATTTCCATGACTCAAATGTGAAGAATCTGTATGAGAAAGTAAAAAGCCAATTAAAGAATAATGCCAAAGAAATCGGAAATGGATGTTTTGAGTTCTACCACAAGTGTGACAATGAATGCATGGAAAGTGTAAGAAATGGGACTTATGATTATCCCAAATATTCAGAAGAGTCAAAGTTGAACAGGGAAAAGGTAGATGGAGTGAAATTGGAATCAATGGGGATCTATCAGGAATTCTCTAGATATCCAGCACAGTGGCGGCCGCTCGCACAAGTAATCAACACTAACAGTCTGTCGCTGCTGACCCAGAATAACCTGAACAAATCCCAGTCCGCACTGGGCACCGCTATCGAGCGTCTGTCTTCTGGTCTGCGTATCAACAGCGCGAAAGACGATGCGGCAGGTCAGGCGATTGCTAACCGTTTCACCGCGAACATCAAAGGTCTGACTCAGGCTTCCCGTAACGCTAACGACGGTATCTCCATTGCGCAGACCACTGAAGGCGCGCTGAACGAAATCAACAACAACCTGCAGCGTGTGCGTGAACTGGCGGTTCAGTCTGCTAACAGCACCAACTCCCAGTCTGACCTCGACTCCATCCAGGCTGAAATCACCCAGCGCCTGAACGAAATCGACCGTGTATCCGGCCAGACTCAGTTCAACGGCGTGAAAGTCCTGGCGCAGGACAACACCCTGACCATCCAGGTTGGCGCCAACGACGGTGAAACTATCGATATCGATCTGAAGCAGATCAACTCTCAGACCCTGGGTCTGGACTCACTGAACGTGCATGGAGCGCCGGTGGATCCTGCTAGCCCATGGACCGAAAACCCGCTGCAGAAAATTGATGCCGCGCTGGCGCAGGTGGATGCGCTGCGCTCTGATCTGGGTGCGGTACAAAACCGTTTCAACTCTGCTATCACCAACCTGGGCAATACCGTAAACAATCTGTCTGAAGCGCGTAGCCGTATCGAAGATTCCGACTACGCGACCGAAGTTTCCAACATGTCTCGCGCGCAGATTTTGCAGCAGGCCGGTACTTCCGTTCTGGCGCAGGCTAACCAGGTCCCGCAGAACGTGCTGTCTCTGTTACGTTAA TAGFOR PRIMER SEQ ID NO: 168 AGGCAAGATCTGACACAATATGTATAGGCTACC REV PRIMERSEQ ID NO: 169 AGTCAGACGCGTCTATTAACGTAACAGAGACAGCAC Ha0s.hisSEQ ID NO: 170GACACAATATGTATAGGCTACCATGCGAACAATTCAACCGACACTGTTGACACAGTACTCGAGAAGAATGTGACAGTGACACACTCTGTTAACCTGCTCGAAGACAGCCACAACGGAAAACTATGTAGATTAAAAGGAATAGCCCCACTACAATTGGGGAAATGTAACATCGCCGGATGGCTTTTGGGAAACCCAGAATGCGACCCACTGCTTCCAGTGAGATCATGGTCCTACATTGTAGAAACACCAAACTCTGAGAATGGAATATGTTATCCAGGAGATTTCATCGACTATGAGGAGCTGAGGGAGCAATTGAGCTCAGTGTCATCATTCGAAAGATTCGAAATATTTCCCAAAGAAAGCTCATGGCCCAACCACAACACAAACGGAGTAACGGCAGCATGCTCCCATGAGGGGAAAAGCAGTTTTTACAGAAATTTGCTATGGCTGACGGAGAAGGAGGGCTCATACCCAAAGCTGAAAAATTCTTATGTGAACAAAAAAGGGAAAGAAGTCCTTGTACTGTGGGGTATTCATCACCCGCCTAACAGTAAGGAACAACAGAATCTCTATCAGAATGAAAATGCTTATGTCTCTGTAGTGACTTCAAATTATAACAGGAGATTTACCCCGGAAATAGCAGAAAGACCCAAAGTAAGAGATCAAGCTGGGAGGATGAACTATTACTGGACCTTGCTAAAACCCGGAGACACAATAATATTTGAGGCAAATGGAAATCTAATAGCACCAATGTATGCTTTCGCACTGAGTAGAGGCTTTGGGTCCGGCATCATCACCTCAAACGCATCAATGCATGAGTGTAACACGAAGTGTCAAACACCCCTGGGAGCTATAAACAGCAGTCTCCCTTACCAGAATATACACCCAGTCACAATAGGAGAGTGCCCAAAATACGTCAGGAGTGCCAAATTGAGGATGGTTACAGGACTAAGGAACATTCCGTCCATTCAATCCAGAGGTCTATTTGGAGCCATTGCCGGTTTTATTGAAGGGGGATGGACTGGAATGATAGATGGATGGTATGGTTATCATCATCAGAATGAACAGGGATCAGGCTATGCAGCGGATCAAAAAAGCACACAAAATGCCATTAACGGGATTACAAACAAGGTGAACACTGTTATCGAGAAAATGAACATTCAATTCACAGCTGTGGGTAAAGAATTCAACAAATTAGAAAAAAGGATGGAAAATTTAAATAAAAAAGTTGATGATGGATTTCTGGACATTTGGACATATAATGCAGAATTGTTAGTTCTACTGGAAAATGAAAGGACTCTGGATTTCCATGACTCAAATGTGAAGAATCTGTATGAGAAAGTAAAAAGCCAATTAAAGAATAATGCCAAAGAAATCGGAAATGGATGTTTTGAGTTCTACCACAAGTGTGACAATGAATGCATGGAAAGTGTAAGAAATGGGACTTATGATTATCCCAAATATTCAGAAGAGTCAAAGTTGAACAGGGAAAAGGTAGATGGAGTGAAATTGGAATCAATGGGGATCTATCAGACGCGTACCGGTCATCATCACCATCAC CATTGAFOR PRIMER SEQ ID NO: 171 ATTCTCCGGCGGCCGCTCGACACAATATGTATAGGCTACCREV PRIMER SEQ ID NO: 172AGTCTTGCGGCCGCCTATTAATGGTGATGGTGATGATGCTGATAGATCCCCATTGATTCC HA0sSEQ ID NO: 173GACACAATATGTATAGGCTACCATGCGAACAATTCAACCGACACTGTTGACACAGTACTCGAGAAGAATGTGACAGTGACACACTCTGTTAACCTGCTCGAAGACAGCCACAACGGAAAACTATGTAGATTAAAAGGAATAGCCCCACTACAATTGGGGAAATGTAACATCGCCGGATGGCTTTTGGGAAACCCAGAATGCGACCCACTGCTTCCAGTGAGATCATGGTCCTACATTGTAGAAACACCAAACTCTGAGAATGGAATATGTTATCCAGGAGATTTCATCGACTATGAGGAGCTGAGGGAGCAATTGAGCTCAGTGTCATCATTCGAAAGATTCGAAATATTTCCCAAAGAAAGCTCATGGCCCAACCACAACACAAACGGAGTAACGGCAGCATGCTCCCATGAGGGGAAAAGCAGTTTTTACAGAAATTTGCTATGGCTGACGGAGAAGGAGGGCTCATACCCAAAGCTGAAAAATTCTTATGTGAACAAAAAAGGGAAAGAAGTCCTTGTACTGTGGGGTATTCATCACCCGCCTAACAGTAAGGAACAACAGAATCTCTATCAGAATGAAAATGCTTATGTCTCTGTAGTGACTTCAAATTATAACAGGAGATTTACCCCGGAAATAGCAGAAAGACCCAAAGTAAGAGATCAAGCTGGGAGGATGAACTATTACTGGACCTTGCTAAAACCCGGAGACACAATAATATTTGAGGCAAATGGAAATCTAATAGCACCAATGTATGCTTTCGCACTGAGTAGAGGCTTTGGGTCCGGCATCATCACCTCAAACGCATCAATGCATGAGTGTAACACGAAGTGTCAAACACCCCTGGGAGCTATAAACAGCAGTCTCCCTTACCAGAATATACACCCAGTCACAATAGGAGAGTGCCCAAAATACGTCAGGAGTGCCAAATTGAGGATGGTTACAGGACTAAGGAACATTCCGTCCATTCAATCCAGAGGTCTATTTGGAGCCATTGCCGGTTTTATTGAAGGGGGATGGACTGGAATGATAGATGGATGGTATGGTTATCATCATCAGAATGAACAGGGATCAGGCTATGCAGCGGATCAAAAAAGCACACAAAATGCCATTAACGGGATTACAAACAAGGTGAACACTGTTATCGAGAAAATGAACATTCAATTCACAGCTGTGGGTAAAGAATTCAACAAATTAGAAAAAAGGATGGAAAATTTAAATAAAAAAGTTGATGATGGATTTCTGGACATTTGGACATATAATGCAGAATTGTTAGTTCTACTGGAAAATGAAAGGACTCTGGATTTCCATGACTCAAATGTGAAGAATCTGTATGAGAAAGTAAAAAGCCAATTAAAGAATAATGCCAAAGAAATCGGAAATGGATGTTTTGAGTTCTACCACAAGTGTGACAATGAATGCATGGAAAGTGTAAGAAATGGGACTTATGATTATCCCAAATATTCAGAAGAGTCAAAGTTGAACAGGGAAAAGGTAGATGGAGTGAAATTGGAATCAATGGGGATCTATCAG FOR PRIMER SEQ ID NO: 174AGGCAAGATCTGACACAATATGTATAGGCTACC REV PRIMER SEQ ID NO: 175AGTCAGACGCGTCTATTAACGTAACAGAGACAGCAC HA0sHis(PR8) SEQ ID NO: 176DTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDSHNGKLCRLKGIAPLQLGKCNIAGWLLGNPECDPLLPVRSWSYIVETPNSENGICYPGDFIDYEELREQLSSVSSFERFEIFPKESSWPNHNTNGVTAACSHEGKSSFYRNLLWLTEKEGSYPKLKNSYVNKKGKEVLVLWGIHHPPNSKEQQNLYQNENAYVSVVTSNYNRRFTPEIAERPKVRDQAGRMNYYWTLLKPGDTIIFEANGNLIAPMYAFALSRGFGSGIITSNASMHECNTKCQTPLGAINSSLPYQNIHPVTIGECPKYVRSAKLRMVTGLRNIPSIQSRGLFGAIAGFIEGGWTGMIDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNTVIEKMNIQFTAVGKEFNKLEKRMENLNKKVDDGFLDIWTYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEIGNGCFEFYHKCDNECMESVRNGTYDYPKYSEESKLNREKVDGVKLESMGIYQH HHHHHSTF2Δ.HA0s(PR8) SEQ ID NO: 177MAQVINTNSLSLLTQNNLNKSQSALGTAIERLSSGLRINSAKDDAAGQAIANRFTANIKGLTQASRNANDGISIAQTTEGALNEINNNLQRVRELAVQSANSTNSQSDLDSIQAEITQRLNEIDRVSGQTQFNGVKVLAQDNTLTIQVGANDGETIDIDLKQINSQTLGLDSLNVHGAPVDPASPWTENPLQKIDAALAQVDALRSDLGAVQNRFNSAITNLGNTVNNLSEARSRIEDSDYATEVSNMSRAQILQQAGTSVLAQANQVPQNVLSLLREFSRYPAQWRPLDTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDSHNGKLCRLKGIAPLQLGKCNIAGWLLGNPECDPLLPVRSWSYIVETPNSENGICYPGDFIDYEELREQLSSVSSFERFEIFPKESSWPNHNTNGVTAACSHEGKSSFYRNLLWLTEKEGSYPKLKNSYVNKKGKEVLVLWGIHHPPNSKEQQNLYQNENAYVSVVTSNYNRRFTPEIAERPKVRDQAGRMNYYWTLLKPGDTIIFEANGNLIAPMYAFALSRGFGSGIITSNASMHECNTKCQTPLGAINSSLPYQNIHPVTIGECPKYVRSAKLRMVTGLRNIPSIQSRGLFGAIAGFIEGGWTGMIDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNTVIEKMNIQFTAVGKEFNKLEKRMENLNKKVDDGFLDIWTYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEIGNGCFEFYHKCDNECMESVRNGTYDYPKYSEESKLNREKVDGVKLESMGIYQ STF2 SEQ ID NO: 178ATGGCACAAGTAATCAACACTAACAGTCTGTCGCTGCTGACCCAGAATAACCTGAACAAATCCCAGTCCGCACTGGGCACCGCTATCGAGCGTCTGTCTTCTGGTCTGCGTATCAACAGCGCGAAAGACGATGCGGCAGGTCAGGCGATTGCTAACCGTTTCACCGCGAACATCAAAGGTCTGACTCAGGCTTCCCGTAACGCTAACGACGGTATCTCCATTGCGCAGACCACTGAAGGCGCGCTGAACGAAATCAACAACAACCTGCAGCGTGTGCGTGAACTGGCGGTTCAGTCTGCTAACAGCACCAACTCCCAGTCTGACCTCGACTCCATCCAGGCTGAAATCACCCAGCGCCTGAACGAAATCGACCGTGTATCCGGCCAGACTCAGTTCAACGGCGTGAAAGTCCTGGCGCAGGACAACACCCTGACCATCCAGGTTGGCGCCAACGACGGTGAAACTATCGATATCGATCTGAAGCAGATCAACTCTCAGACCCTGGGTCTGGACTCACTGAACGTGCAGAAAGCGTATGATGTGAAAGATACAGCAGTAACAACGAAAGCTTATGCCAATAATGGTACTACACTGGATGTATCGGGTCTTGATGATGCAGCTATTAAAGCGGCTACGGGTGGTACGAATGGTACGGCTTCTGTAACCGGTGGTGCGGTTAAATTTGACGCAGATAATAACAAGTACTTTGTTACTATTGGTGGCTTTACTGGTGCTGATGCCGCCAAAAATGGCGATTATGAAGTTAACGTTGCTACTGACGGTACAGTAACCCTTGCGGCTGGCGCAACTAAAACCACAATGCCTGCTGGTGCGACAACTAAAACAGAAGTACAGGAGTTAAAAGATACACCGGCAGTTGTTTCAGCAGATGCTAAAAATGCCTTAATTGCTGGCGGCGTTGACGCTACCGATGCTAATGGCGCTGAGTTGGTCAAAATGTCTTATACCGATAAAAATGGTAAGACAATTGAAGGCGGTTATGCGCTTAAAGCTGGCGATAAGTATTACGCCGCAGATTACGATGAAGCGACAGGAGCAATTAAAGCTAAAACTACAAGTTATACTGCTGCTGACGGCACTACCAAAACAGCGGCTAACCAACTGGGTGGCGTAGACGGTAAAACCGAAGTCGTTACTATCGACGGTAAAACCTACAATGCCAGCAAAGCCGCTGGTCATGATTTCAAAGCACAACCAGAGCTGGCGGAAGCAGCCGCTAAAACCACCGAAAACCCGCTGCAGAAAATTGATGCCGCGCTGGCGCAGGTGGATGCGCTGCGCTCTGATCTGGGTGCGGTACAAAACCGTTTCAACTCTGCTATCACCAACCTGGGCAATACCGTAAACAATCTGTCTGAAGCGCGTAGCCGTATCGAAGATTCCGACTACGCGACCGAAGTTTCCAACATGTCTCGCGCGCAGATTCTGCAGCAGGCCGGTACTTCCGTTCTGGCGCAGGCTAACCAGGTCCCGCAGAACGTGCTGTCTCTGTTACGT HA1-1his(PR8) SEQ ID No: 179MKFLVNVALVFMVVYISYIYASHNGKLCRLKGIAPLQLGKCNIAGWLLGNPECDPLLPVRSWSYIVETPNSENGICYPGDFIDYEELREQLSSVSSFERFEIFPKESSWPNHNTNGVTAACSHEGKSSFYRNLLWLTEKEGSYPKLKNSYVNKKGKEVLVLWGIHHPPNSKEQQNLYQNENAYVSVVTSNYNRRFTPEIAERPKVRDQAGRMNYYWTLLKPGDTIIFEANGNLIAPMYAFALSRGFGSGIITSNASMHECNTKCQTPLGAINSSLPYQNIHPVTIGECPKYVRHHHHHH HA1-1his(VN)SEQ ID No: 180MKFLVNVALVFMVVYISYIYAEKKHNGKLCDLDGVKPLILRDCSVAGWLLGNPMCDEFINVPEWSYIVEKANPVNDLCYPGDFNDYEELKHLLSRINHFEKIQIIPKSSWSSHEASLGVSSACPYQGKSSFFRNVVWLIKKNSTYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQTKLYQNPTTYISVGTSTLNQRLVPRIATRSKVNGQSGRMEFFWTILKPNDAINFESNGNFIAPEYAYKIVKKGDSTIMKSELEYGNCNTKCQTPMGAINSSMPFHNIHPLTIGECPKYVKHHHHH HHA1-1his(IND) SEQ ID No: 181MKFLVNVALVFMVVYISYIYAEKTHNGKLCDLDGVKPLILRDCSVAGWLLGNPMCDEFINVPEWSYIVEKANPTNDLCYPGSFNDYEELKHLLSRINHFEKIQIIPKSSWSDHEASSGVSSACPYLGSPSFFRNVVWLIKKNSTYPTIKKSYNNTNQEDLLVLWGIHHPNDAAEQTRLYQNPTTYISIGTSTLNQRLVPKIATRSKVNGQSGRMEFFWTILKPNDAINFESNGNFIAPEYAYKIVKKGDSAIMKSELEYGNCNTKCQTPMGAINSSMPFHNIHPLTIGECPKYVKHHHHH HHA1-1his(NC) SEQ ID No: 182MKFLVNVALVFMVVYISYIYASHNGKLCLLKGIAPLQLGNCSVAGWILGNPECELLISKESWSYIVETPNPENGTCYPGYFADYEELREQLSSVSSFERFEIFPKESSWPNHTVTGVSASCSHNGKSSFYRNLLWLTGKNGLYPNLSKSYVNNKEKEVLVLWGVHHPPNIGNQRALYHTENAYVSVVSSHYSRRFTPEIAKRPKVRDQEGRINYYWTLLEPGDTIIFEANGNLIAPWYAFALSRGFGSGIITSNAPMDECDAKCQTPQGAINSSLPFQNVHPVTIGECPKYVRHHHHHH HA1-1(MAL)SEQ ID No: 183ACCACTACCCCAACCAAATCTCACTTCGCAAACCTGAAAGGCACTGAAACCCGTGGCAAGCTGTGTCCGAAGTGTCTGAACTGCACCGATCTGGACGTCGCACTGGGTCGTCCGAAATGTACTGGTAACATTCCGTCCGCGCGTGTCTCCATCCTGCATGAAGTGCGTCCAGTGACCTCCGGCTGTTTTCCGATTATGCATGATCGTACTAAAATCCGTCAGCTGCCGAACCTGCTGCGTGGTTACGAACACATTCGTCTGTCCACCCATAACGTTATCAACGCGGAAAACGCGCCGGGCGGTAGCTATAAAATCGGTACCTCTGGTTCTTGCCCGAACGTGACTAACGGTAACGGCTTCTTTGCAACCATGGCCTGGGCGGTCCCGAAAAACGACAACAACAAGACCGCGACCAATTCCCTGACCATCGAAGTCCCGTATATCTGCACCGAAGGTGAAGATCAAATCACGGTTTGGGGCTTCCACTCCGACAACGAGGCACAAATGGCGAAACTGTACGGTGACAGCAAACCGCAAAAATTCACTAGCTCCGCTAACGGTGTTACCACCCACTACGTTTCCCAGATCGGTGGTTTCCCAAACCAGACCGAAGATGGTGGTCTGCCGCAGTCCGGTCGCATCGTTGTAGATTATATGGTGCAGAAAAGCGGTAAAACCGGTACCATCACCTACCAGCGTGGCATCCTGCTGCCGCAGAAAGTTTGGTGCGCTTCCGGTCGTAGCAAAGTAATCAAAGGTTCCCTGCCGCTGATCGGTGAAGCAGACTGCCTGCACGAGAAATACGGCGGTCTGAACAAAAGCAAGCCGTACTATACCGGCGAACATGCGAAAGCAATTGGTAACTGTCCAATTTGGGTGAAATAGTAG STF2.HA1-1(MAL)SEQ ID No: 184ATGGCACAAGTAATCAACACTAACAGTCTGTCGCTGCTGACCCAGAATAACCTGAACAAATCCCAGTCCGCACTGGGCACCGCTATCGAGCGTCTGTCTTCTGGTCTGCGTATCAACAGCGCGAAAGACGATGCGGCAGGTCAGGCGATTGCTAACCGTTTCACCGCGAACATCAAAGGTCTGACTCAGGCTTCCCGTAACGCTAACGACGGTATCTCCATTGCGCAGACCACTGAAGGCGCGCTGAACGAAATCAACAACAACCTGCAGCGTGTGCGTGAACTGGCGGTTCAGTCTGCTAACAGCACCAACTCCCAGTCTGACCTCGACTCCATCCAGGCTGAAATCACCCAGCGCCTGAACGAAATCGACCGTGTATCCGGCCAGACTCAGTTCAACGGCGTGAAAGTCCTGGCGCAGGACAACACCCTGACCATCCAGGTTGGCGCCAACGACGGTGAAACTATCGATATCGATCTGAAGCAGATCAACTCTCAGACCCTGGGTCTGGACTCACTGAACGTGCAGAAAGCGTATGATGTGAAAGATACAGCAGTAACAACGAAAGCTTATGCCAATAATGGTACTACACTGGATGTATCGGGTCTTGATGATGCAGCTATTAAAGCGGCTACGGGTGGTACGAATGGTACGGCTTCTGTAACCGGTGGTGCGGTTAAATTTGACGCAGATAATAACAAGTACTTTGTTACTATTGGTGGCTTTACTGGTGCTGATGCCGCCAAAAATGGCGATTATGAAGTTAACGTTGCTACTGACGGTACAGTAACCCTTGCGGCTGGCGCAACTAAAACCACAATGCCTGCTGGTGCGACAACTAAAACAGAAGTACAGGAGTTAAAAGATACACCGGCAGTTGTTTCAGCAGATGCTAAAAATGCCTTAATTGCTGGCGGCGTTGACGCTACCGATGCTAATGGCGCTGAGTTGGTCAAAATGTCTTATACCGATAAAAATGGTAAGACAATTGAAGGCGGTTATGCGCTTAAAGCTGGCGATAAGTATTACGCCGCAGATTACGATGAAGCGACAGGAGCAATTAAAGCTAAAACTACAAGTTATACTGCTGCTGACGGCACTACCAAAACAGCGGCTAACCAACTGGGTGGCGTAGACGGTAAAACCGAAGTCGTTACTATCGACGGTAAAACCTACAATGCCAGCAAAGCCGCTGGTCATGATTTCAAAGCACAACCAGAGCTGGCGGAAGCAGCCGCTAAAACCACCGAAAACCCGCTGCAGAAAATTGATGCCGCGCTGGCGCAGGTGGATGCGCTGCGCTCTGATCTGGGTGCGGTACAAAACCGTTTCAACTCTGCTATCACCAACCTGGGCAATACCGTAAACAATCTGTCTGAAGCGCGTAGCCGTATCGAAGATTCCGACTACGCGACCGAAGTTTCCAACATGTCTCGCGCGCAGATTCTGCAGCAGGCCGGTACTTCCGTTCTGGCGCAGGCTAACCAGGTCCCGCAGAACGTGCTGTCTCTGTTAGCGACCACTACCCCAACCAAATCTCACTTCGCAAACCTGAAAGGCACTGAAACCCGTGGCAAGCTGTGTCCGAAGTGTCTGAACTGCACCGATCTGGACGTCGCACTGGGTCGTCCGAAATGTACTGGTAACATTCCGTCCGCGCGTGTCTCCATCCTGCATGAAGTGCGTCCAGTGACCTCCGGCTGTTTTCCGATTATGCATGATCGTACTAAAATCCGTCAGCTGCCGAACCTGCTGCGTGGTTACGAACACATTCGTCTGTCCACCCATAACGTTATCAACGCGGAAAACGCGCCGGGCGGTAGCTATAAAATCGGTACCTCTGGTTCTTGCCCGAACGTGACTAACGGTAACGGCTTCTTTGCAACCATGGCCTGGGCGGTCCCGAAAAACGACAACAACAAGACCGCGACCAATTCCCTGACCATCGAAGTCCCGTATATCTGCACCGAAGGTGAAGATCAAATCACGGTTTGGGGCTTCCACTCCGACAACGAGGCACAAATGGCGAAACTGTACGGTGACAGCAAACCGCAAAAATTCACTAGCTCCGCTAACGGTGTTACCACCCACTACGTTTCCCAGATCGGTGGTTTCCCAAACCAGACCGAAGATGGTGGTCTGCCGCAGTCCGGTCGCATCGTTGTAGATTATATGGTGCAGAAAAGCGGTAAAACCGGTACCATCACCTACCAGCGTGGCATCCTGCTGCCGCAGAAAGTTTGGTGCGCTTCCGGTCGTAGCAAAGTAATCAAAGGTTCCCTGCCGCTGATCGGTGAAGCAGACTGCCTGCACGAGAAATACGGCGGTCTGAACAAAAGCAAGCCGTACTATACCGGCGAACATGCGAAAGCAATTGGTAACTGTCCAATTTGGGTGAAA TAGTAGHA1-2(MAL) SEQ ID No: 185AAAGGCACTGAAACCCGTGGCAAGCTGTGTCCGAAGTGTCTGAACTGCACCGATCTGGACGTCGCACTGGGTCGTCCGAAATGTACTGGTAACATTCCGTCCGCGCGTGTCTCCATCCTGCATGAAGTGCGTCCAGTGACCTCCGGCTGTTTTCCGATTATGCATGATCGTACTAAAATCCGTCAGCTGCCGAACCTGCTGCGTGGTTACGAACACATTCGTCTGTCCACCCATAACGTTATCAACGCGGAAAACGCGCCGGGCGGTAGCTATAAAATCGGTACCTCTGGTTCTTGCCCGAACGTGACTAACGGTAACGGCTTCTTTGCAACCATGGCCTGGGCGGTCCCGAAAAACGACAACAACAAGACCGCGACCAATTCCCTGACCATCGAAGTCCCGTATATCTGCACCGAAGGTGAAGATCAAATCACGGTTTGGGGCTTCCACTCCGACAACGAGGCACAAATGGCGAAACTGTACGGTGACAGCAAACCGCAAAAATTCACTAGCTCCGCTAACGGTGTTACCACCCACTACGTTTCCCAGATCGGTGGTTTCCCAAACCAGACCGAAGATGGTGGTCTGCCGCAGTCCGGTCGCATCGTTGTAGATTATATGGTGCAGAAAAGCGGTAAAACCGGTACCATCACCTACCAGCGTGGCATCCTGCTGCCGCAGAAAGTTTGGTGCGCTTCCGGTCGTAGCAAAGTAATCAAA GGTTGATAGSTF2.HA1-2(MAL) SEQ ID No: 186ATGGCACAAGTAATCAACACTAACAGTCTGTCGCTGCTGACCCAGAATAACCTGAACAAATCCCAGTCCGCACTGGGCACCGCTATCGAGCGTCTGTCTTCTGGTCTGCGTATCAACAGCGCGAAAGACGATGCGGCAGGTCAGGCGATTGCTAACCGTTTCACCGCGAACATCAAAGGTCTGACTCAGGCTTCCCGTAACGCTAACGACGGTATCTCCATTGCGCAGACCACTGAAGGCGCGCTGAACGAAATCAACAACAACCTGCAGCGTGTGCGTGAACTGGCGGTTCAGTCTGCTAACAGCACCAACTCCCAGTCTGACCTCGACTCCATCCAGGCTGAAATCACCCAGCGCCTGAACGAAATCGACCGTGTATCCGGCCAGACTCAGTTCAACGGCGTGAAAGTCCTGGCGCAGGACAACACCCTGACCATCCAGGTTGGCGCCAACGACGGTGAAACTATCGATATCGATCTGAAGCAGATCAACTCTCAGACCCTGGGTCTGGACTCACTGAACGTGCAGAAAGCGTATGATGTGAAAGATACAGCAGTAACAACGAAAGCTTATGCCAATAATGGTACTACACTGGATGTATCGGGTCTTGATGATGCAGCTATTAAAGCGGCTACGGGTGGTACGAATGGTACGGCTTCTGTAACCGGTGGTGCGGTTAAATTTGACGCAGATAATAACAAGTACTTTGTTACTATTGGTGGCTTTACTGGTGCTGATGCCGCCAAAAATGGCGATTATGAAGTTAACGTTGCTACTGACGGTACAGTAACCCTTGCGGCTGGCGCAACTAAAACCACAATGCCTGCTGGTGCGACAACTAAAACAGAAGTACAGGAGTTAAAAGATACACCGGCAGTTGTTTCAGCAGATGCTAAAAATGCCTTAATTGCTGGCGGCGTTGACGCTACCGATGCTAATGGCGCTGAGTTGGTCAAAATGTCTTATACCGATAAAAATGGTAAGACAATTGAAGGCGGTTATGCGCTTAAAGCTGGCGATAAGTATTACGCCGCAGATTACGATGAAGCGACAGGAGCAATTAAAGCTAAAACTACAAGTTATACTGCTGCTGACGGCACTACCAAAACAGCGGCTAACCAACTGGGTGGCGTAGACGGTAAAACCGAAGTCGTTACTATCGACGGTAAAACCTACAATGCCAGCAAAGCCGCTGGTCATGATTTCAAAGCACAACCAGAGCTGGCGGAAGCAGCCGCTAAAACCACCGAAAACCCGCTGCAGAAAATTGATGCCGCGCTGGCGCAGGTGGATGCGCTGCGCTCTGATCTGGGTGCGGTACAAAACCGTTTCAACTCTGCTATCACCAACCTGGGCAATACCGTAAACAATCTGTCTGAAGCGCGTAGCCGTATCGAAGATTCCGACTACGCGACCGAAGTTTCCAACATGTCTCGCGCGCAGATTCTGCAGCAGGCCGGTACTTCCGTTCTGGCGCAGGCTAACCAGGTCCCGCAGAACGTGCTGTCTCTGTTAGCGAAAGGCACTGAAACCCGTGGCAAGCTGTGTCCGAAGTGTCTGAACTGCACCGATCTGGACGTCGCACTGGGTCGTCCGAAATGTACTGGTAACATTCCGTCCGCGCGTGTCTCCATCCTGCATGAAGTGCGTCCAGTGACCTCCGGCTGTTTTCCGATTATGCATGATCGTACTAAAATCCGTCAGCTGCCGAACCTGCTGCGTGGTTACGAACACATTCGTCTGTCCACCCATAACGTTATCAACGCGGAAAACGCGCCGGGCGGTAGCTATAAAATCGGTACCTCTGGTTCTTGCCCGAACGTGACTAACGGTAACGGCTTCTTTGCAACCATGGCCTGGGCGGTCCCGAAAAACGACAACAACAAGACCGCGACCAATTCCCTGACCATCGAAGTCCCGTATATCTGCACCGAAGGTGAAGATCAAATCACGGTTTGGGGCTTCCACTCCGACAACGAGGCACAAATGGCGAAACTGTACGGTGACAGCAAACCGCAAAAATTCACTAGCTCCGCTAACGGTGTTACCACCCACTACGTTTCCCAGATCGGTGGTTTCCCAAACCAGACCGAAGATGGTGGTCTGCCGCAGTCCGGTCGCATCGTTGTAGATTATATGGTGCAGAAAAGCGGTAAAACCGGTACCATCACCTACCAGCGTGGCATCCTGCTGCCGCAGAAAGTTTGGTGCGCTTCCGGTCGTAGCAAAGTAATCAAAGGTTGATAG HA1-2(SH) SEQ ID No: 187AAAGGCACTCGTACCCGCGGTAAGCTGTGCCCGGACTGCCTGAACTGTACCGATCTGGATGTTGCACTGGGTCGTCCGATGTGCGTTGGTACCACCCCGTCTGCGAAAGCCAGCATCCTGCACGAAGTTCGCCCGGTTACTTCCGGTTGTTTCCCGATTATGCATGATCGTACCAAAATTCGTCAGCTGCCAAACCTGCTGCGTGGCTATGAAAACATTCGTCTGTCCACTCAAAACGTAATCGATGCAGAAAAAGCGCTGGGTGGCCCGTATCGTCTGGGTACCAGCGGCTCCTGCCCGAACGCGACGAGCAAAAGCGGCTTCTTCGCCACCATGGCTTGGGCCGTTCCGAAAGACAACAACAAAAACGCTACGAACCCGCTGACCGTCGAAGTCCCGTACATCTGCACCGAAGGCGAAGATCAGATCACTGTGTGGGGCTTCCACAGCGATGATAAGACCCAGATGAAAAATCTGTACGGTGACTCCAACCCGCAGAAATTCACCTCTTCTGCTAACGGTGTAACGACCCACTACGTTTCTCAGATCGGTGGTTTCCCGGACCAGACGGAAGATGGCGGTCTGCCTCAGTCCGGCCGCATCGTAGTTGATTACATGGTCCAGAAACCGGGTAAGACTGGTACCATTGTTTACCAGCGTGGTGTACTGCTGCCGCAGAAGGTCTGGTGTGCTTCCGGCCGTTCCAAGGTCATTAAGGGC TGATASTF2.HA1-2(SH) SEQ ID No: 188ATGGCACAAGTAATCAACACTAACAGTCTGTCGCTGCTGACCCAGAATAACCTGAACAAATCCCAGTCCGCACTGGGCACCGCTATCGAGCGTCTGTCTTCTGGTCTGCGTATCAACAGCGCGAAAGACGATGCGGCAGGTCAGGCGATTGCTAACCGTTTCACCGCGAACATCAAAGGTCTGACTCAGGCTTCCCGTAACGCTAACGACGGTATCTCCATTGCGCAGACCACTGAAGGCGCGCTGAACGAAATCAACAACAACCTGCAGCGTGTGCGTGAACTGGCGGTTCAGTCTGCTAACAGCACCAACTCCCAGTCTGACCTCGACTCCATCCAGGCTGAAATCACCCAGCGCCTGAACGAAATCGACCGTGTATCCGGCCAGACTCAGTTCAACGGCGTGAAAGTCCTGGCGCAGGACAACACCCTGACCATCCAGGTTGGCGCCAACGACGGTGAAACTATCGATATCGATCTGAAGCAGATCAACTCTCAGACCCTGGGTCTGGACTCACTGAACGTGCAGAAAGCGTATGATGTGAAAGATACAGCAGTAACAACGAAAGCTTATGCCAATAATGGTACTACACTGGATGTATCGGGTCTTGATGATGCAGCTATTAAAGCGGCTACGGGTGGTACGAATGGTACGGCTTCTGTAACCGGTGGTGCGGTTAAATTTGACGCAGATAATAACAAGTACTTTGTTACTATTGGTGGCTTTACTGGTGCTGATGCCGCCAAAAATGGCGATTATGAAGTTAACGTTGCTACTGACGGTACAGTAACCCTTGCGGCTGGCGCAACTAAAACCACAATGCCTGCTGGTGCGACAACTAAAACAGAAGTACAGGAGTTAAAAGATACACCGGCAGTTGTTTCAGCAGATGCTAAAAATGCCTTAATTGCTGGCGGCGTTGACGCTACCGATGCTAATGGCGCTGAGTTGGTCAAAATGTCTTATACCGATAAAAATGGTAAGACAATTGAAGGCGGTTATGCGCTTAAAGCTGGCGATAAGTATTACGCCGCAGATTACGATGAAGCGACAGGAGCAATTAAAGCTAAAACTACAAGTTATACTGCTGCTGACGGCACTACCAAAACAGCGGCTAACCAACTGGGTGGCGTAGACGGTAAAACCGAAGTCGTTACTATCGACGGTAAAACCTACAATGCCAGCAAAGCCGCTGGTCATGATTTCAAAGCACAACCAGAGCTGGCGGAAGCAGCCGCTAAAACCACCGAAAACCCGCTGCAGAAAATTGATGCCGCGCTGGCGCAGGTGGATGCGCTGCGCTCTGATCTGGGTGCGGTACAAAACCGTTTCAACTCTGCTATCACCAACCTGGGCAATACCGTAAACAATCTGTCTGAAGCGCGTAGCCGTATCGAAGATTCCGACTACGCGACCGAAGTTTCCAACATGTCTCGCGCGCAGATTCTGCAGCAGGCCGGTACTTCCGTTCTGGCGCAGGCTAACCAGGTCCCGCAGAACGTGCTGTCTCTGTTAGCGAAAGGCACTCGTACCCGCGGTAAGCTGTGCCCGGACTGCCTGAACTGTACCGATCTGGATGTTGCACTGGGTCGTCCGATGTGCGTTGGTACCACCCCGTCTGCGAAAGCCAGCATCCTGCACGAAGTTCGCCCGGTTACTTCCGGTTGTTTCCCGATTATGCATGATCGTACCAAAATTCGTCAGCTGCCAAACCTGCTGCGTGGCTATGAAAACATTCGTCTGTCCACTCAAAACGTAATCGATGCAGAAAAAGCGCTGGGTGGCCCGTATCGTCTGGGTACCAGCGGCTCCTGCCCGAACGCGACGAGCAAAAGCGGCTTCTTCGCCACCATGGCTTGGGCCGTTCCGAAAGACAACAACAAAAACGCTACGAACCCGCTGACCGTCGAAGTCCCGTACATCTGCACCGAAGGCGAAGATCAGATCACTGTGTGGGGCTTCCACAGCGATGATAAGACCCAGATGAAAAATCTGTACGGTGACTCCAACCCGCAGAAATTCACCTCTTCTGCTAACGGTGTAACGACCCACTACGTTTCTCAGATCGGTGGTTTCCCGGACCAGACGGAAGATGGCGGTCTGCCTCAGTCCGGCCGCATCGTAGTTGATTACATGGTCCAGAAACCGGGTAAGACTGGTACCATTGTTTACCAGCGTGGTGTACTGCTGCCGCAGAAGGTCTGGTGTGCTTCCGGCCGTTCCAAGGTCATTAAGGGCTGATAG HA1-2(Lee) SEQ ID No: 189AAAGGCACTCAGACCCGTGGCAAGCTGTGTCCGAACTGTTTCAACTGCACCGATCTGGACGTTGCACTGGGTCGTCCGAAATGCATGGGTAACATCCCGTCTGCGAAGGTAAGCATCCTGCACGAAGTTAAACCGGTAACCAGCGGCTGTTTCCCGATCATGCACGACAAAACTAAAATTCGTCAGCTGCCGAACCTGCTGCGTGGTTATGAGAACATTCGTCTGTCTACCTCTAATGTTATCAACGCGGAGACTGCACCAGGTGGCCCATACAAAGTAGGTACCAGCGGTTCCTGTCCGAACGTTGCGAATCGTAACGGCTTCTTCAACACTATGGCGTGGGTTATCCCGAAAGATAACAATAAAACTGCAATTAACCCGGTAACTGTAGAAGTTCCGTACATCTGCTCCGAAGGCGAGGACCAGATTACGGTATGGGGCTTTCACAGCGACGATAAAACCCAGATGGAGCGTCTGTACGGTGACTCTAACCCGCAGAAATTCACCTCCTCCGCGAACGGCGTTACCACCCACTATGTTTCTCAGATCGGCGGTTTCCCGAATCAGACCGAAGACGAAGGCCTGAAGCAGTCCGGCCGTATTGTTGTAGACTACATGGTTCAGAAGCCGGGCAAAACTGGTACCATTGTATACCAGCGCGGCATCCTGCTGCCGCAGAAAGTTTGGTGCGCTTCCGGTCGTAGCAAAGTAATCAAAGGT TGATAGSTF2.HA1-2(Lee) SEQ ID No: 190ATGGCACAAGTAATCAACACTAACAGTCTGTCGCTGCTGACCCAGAATAACCTGAACAAATCCCAGTCCGCACTGGGCACCGCTATCGAGCGTCTGTCTTCTGGTCTGCGTATCAACAGCGCGAAAGACGATGCGGCAGGTCAGGCGATTGCTAACCGTTTCACCGCGAACATCAAAGGTCTGACTCAGGCTTCCCGTAACGCTAACGACGGTATCTCCATTGCGCAGACCACTGAAGGCGCGCTGAACGAAATCAACAACAACCTGCAGCGTGTGCGTGAACTGGCGGTTCAGTCTGCTAACAGCACCAACTCCCAGTCTGACCTCGACTCCATCCAGGCTGAAATCACCCAGCGCCTGAACGAAATCGACCGTGTATCCGGCCAGACTCAGTTCAACGGCGTGAAAGTCCTGGCGCAGGACAACACCCTGACCATCCAGGTTGGCGCCAACGACGGTGAAACTATCGATATCGATCTGAAGCAGATCAACTCTCAGACCCTGGGTCTGGACTCACTGAACGTGCAGAAAGCGTATGATGTGAAAGATACAGCAGTAACAACGAAAGCTTATGCCAATAATGGTACTACACTGGATGTATCGGGTCTTGATGATGCAGCTATTAAAGCGGCTACGGGTGGTACGAATGGTACGGCTTCTGTAACCGGTGGTGCGGTTAAATTTGACGCAGATAATAACAAGTACTTTGTTACTATTGGTGGCTTTACTGGTGCTGATGCCGCCAAAAATGGCGATTATGAAGTTAACGTTGCTACTGACGGTACAGTAACCCTTGCGGCTGGCGCAACTAAAACCACAATGCCTGCTGGTGCGACAACTAAAACAGAAGTACAGGAGTTAAAAGATACACCGGCAGTTGTTTCAGCAGATGCTAAAAATGCCTTAATTGCTGGCGGCGTTGACGCTACCGATGCTAATGGCGCTGAGTTGGTCAAAATGTCTTATACCGATAAAAATGGTAAGACAATTGAAGGCGGTTATGCGCTTAAAGCTGGCGATAAGTATTACGCCGCAGATTACGATGAAGCGACAGGAGCAATTAAAGCTAAAACTACAAGTTATACTGCTGCTGACGGCACTACCAAAACAGCGGCTAACCAACTGGGTGGCGTAGACGGTAAAACCGAAGTCGTTACTATCGACGGTAAAACCTACAATGCCAGCAAAGCCGCTGGTCATGATTTCAAAGCACAACCAGAGCTGGCGGAAGCAGCCGCTAAAACCACCGAAAACCCGCTGCAGAAAATTGATGCCGCGCTGGCGCAGGTGGATGCGCTGCGCTCTGATCTGGGTGCGGTACAAAACCGTTTCAACTCTGCTATCACCAACCTGGGCAATACCGTAAACAATCTGTCTGAAGCGCGTAGCCGTATCGAAGATTCCGACTACGCGACCGAAGTTTCCAACATGTCTCGCGCGCAGATTCTGCAGCAGGCCGGTACTTCCGTTCTGGCGCAGGCTAACCAGGTCCCGCAGAACGTGCTGTCTCTGTTAGCGAAAGGCACTCAGACCCGTGGCAAGCTGTGTCCGAACTGTTTCAACTGCACCGATCTGGACGTTGCACTGGGTCGTCCGAAATGCATGGGTAACATCCCGTCTGCGAAGGTAAGCATCCTGCACGAAGTTAAACCGGTAACCAGCGGCTGTTTCCCGATCATGCACGACAAAACTAAAATTCGTCAGCTGCCGAACCTGCTGCGTGGTTATGAGAACATTCGTCTGTCTACCTCTAATGTTATCAACGCGGAGACTGCACCAGGTGGCCCATACAAAGTAGGTACCAGCGGTTCCTGTCCGAACGTTGCGAATCGTAACGGCTTCTTCAACACTATGGCGTGGGTTATCCCGAAAGATAACAATAAAACTGCAATTAACCCGGTAACTGTAGAAGTTCCGTACATCTGCTCCGAAGGCGAGGACCAGATTACGGTATGGGGCTTTCACAGCGACGATAAAACCCAGATGGAGCGTCTGTACGGTGACTCTAACCCGCAGAAATTCACCTCCTCCGCGAACGGCGTTACCACCCACTATGTTTCTCAGATCGGCGGTTTCCCGAATCAGACCGAAGACGAAGGCCTGAAGCAGTCCGGCCGTATTGTTGTAGACTACATGGTTCAGAAGCCGGGCAAAACTGGTACCATTGTATACCAGCGCGGCATCCTGCTGCCGCAGAAAGTTTGGTGCGCTTCCGGTCGTAGCAAAGTAATCAAAGGTTGATAG HA1-2(Ohio) SEQ ID No: 191AAAGGCACTAAAACCCGTGGCAAGCTGTGTCCGAAGTGTCTGAACTGCACCGATCTGGACGTCGCACTGGGTCGTCCGAAATGTACTGGTAACATTCCGTCCGCGGAAGTCTCCATCCTGCATGAAGTGCGTCCAGTGACCTCCGGCTGTTTTCCGATTATGCATGATCGTACTAAAATCCGTCAGCTGCCGAACCTGCTGCGTGGTTACGAACACATTCGTCTGTCCACCCATAACGTTATCAACGCGGAAAAGGCGCCGGGCGGTCCCTATAAAATCGGTACCTCTGGTTCTTGCCCGAACGTGACTAACGGTAACGGCTTCTTTGCAACCATGGCCTGGGCGGTCCCGAAAAACGACAACAACAAGACCGCGACCAATTCCCTGACCATCGAAGTCCCGTATATCTGCACCGAAGGTGAAGATCAAATCACGATTTGGGGCTTCCACTCCGACAGCGAGACACAAATGGCGAAACTGTACGGTGACAGCAAACCGCAAAAATTCACTAGCTCCGCTAACGGTGTTACCACCCACTACGTTTCCCAGATCGGTGGTTTCCCAAACCAGACCGAAGATGGTGGTCTGCCGCAGTCCGGTCGCATCGTTGTAGATTATATGGTGCAGAAAAGCGGTAAAACCGGTACCATCACCTACCAGCGTGGCATCCTGCTGCCGCAGAAAGTTTGGTGCGCTTCCGGTCGTAGCAAAGTAATCAAA GGTTGATAGSTF2.HA1-2(Ohio) SEQ ID No: 192ATGGCACAAGTAATCAACACTAACAGTCTGTCGCTGCTGACCCAGAATAACCTGAACAAATCCCAGTCCGCACTGGGCACCGCTATCGAGCGTCTGTCTTCTGGTCTGCGTATCAACAGCGCGAAAGACGATGCGGCAGGTCAGGCGATTGCTAACCGTTTCACCGCGAACATCAAAGGTCTGACTCAGGCTTCCCGTAACGCTAACGACGGTATCTCCATTGCGCAGACCACTGAAGGCGCGCTGAACGAAATCAACAACAACCTGCAGCGTGTGCGTGAACTGGCGGTTCAGTCTGCTAACAGCACCAACTCCCAGTCTGACCTCGACTCCATCCAGGCTGAAATCACCCAGCGCCTGAACGAAATCGACCGTGTATCCGGCCAGACTCAGTTCAACGGCGTGAAAGTCCTGGCGCAGGACAACACCCTGACCATCCAGGTTGGCGCCAACGACGGTGAAACTATCGATATCGATCTGAAGCAGATCAACTCTCAGACCCTGGGTCTGGACTCACTGAACGTGCAGAAAGCGTATGATGTGAAAGATACAGCAGTAACAACGAAAGCTTATGCCAATAATGGTACTACACTGGATGTATCGGGTCTTGATGATGCAGCTATTAAAGCGGCTACGGGTGGTACGAATGGTACGGCTTCTGTAACCGGTGGTGCGGTTAAATTTGACGCAGATAATAACAAGTACTTTGTTACTATTGGTGGCTTTACTGGTGCTGATGCCGCCAAAAATGGCGATTATGAAGTTAACGTTGCTACTGACGGTACAGTAACCCTTGCGGCTGGCGCAACTAAAACCACAATGCCTGCTGGTGCGACAACTAAAACAGAAGTACAGGAGTTAAAAGATACACCGGCAGTTGTTTCAGCAGATGCTAAAAATGCCTTAATTGCTGGCGGCGTTGACGCTACCGATGCTAATGGCGCTGAGTTGGTCAAAATGTCTTATACCGATAAAAATGGTAAGACAATTGAAGGCGGTTATGCGCTTAAAGCTGGCGATAAGTATTACGCCGCAGATTACGATGAAGCGACAGGAGCAATTAAAGCTAAAACTACAAGTTATACTGCTGCTGACGGCACTACCAAAACAGCGGCTAACCAACTGGGTGGCGTAGACGGTAAAACCGAAGTCGTTACTATCGACGGTAAAACCTACAATGCCAGCAAAGCCGCTGGTCATGATTTCAAAGCACAACCAGAGCTGGCGGAAGCAGCCGCTAAAACCACCGAAAACCCGCTGCAGAAAATTGATGCCGCGCTGGCGCAGGTGGATGCGCTGCGCTCTGATCTGGGTGCGGTACAAAACCGTTTCAACTCTGCTATCACCAACCTGGGCAATACCGTAAACAATCTGTCTGAAGCGCGTAGCCGTATCGAAGATTCCGACTACGCGACCGAAGTTTCCAACATGTCTCGCGCGCAGATTCTGCAGCAGGCCGGTACTTCCGTTCTGGCGCAGGCTAACCAGGTCCCGCAGAACGTGCTGTCTCTGTTAGCGAAAGGCACTAAAACCCGTGGCAAGCTGTGTCCGAAGTGTCTGAACTGCACCGATCTGGACGTCGCACTGGGTCGTCCGAAATGTACTGGTAACATTCCGTCCGCGGAAGTCTCCATCCTGCATGAAGTGCGTCCAGTGACCTCCGGCTGTTTTCCGATTATGCATGATCGTACTAAAATCCGTCAGCTGCCGAACCTGCTGCGTGGTTACGAACACATTCGTCTGTCCACCCATAACGTTATCAACGCGGAAAAGGCGCCGGGCGGTCCCTATAAAATCGGTACCTCTGGTTCTTGCCCGAACGTGACTAACGGTAACGGCTTCTTTGCAACCATGGCCTGGGCGGTCCCGAAAAACGACAACAACAAGACCGCGACCAATTCCCTGACCATCGAAGTCCCGTATATCTGCACCGAAGGTGAAGATCAAATCACGATTTGGGGCTTCCACTCCGACAGCGAGACACAAATGGCGAAACTGTACGGTGACAGCAAACCGCAAAAATTCACTAGCTCCGCTAACGGTGTTACCACCCACTACGTTTCCCAGATCGGTGGTTTCCCAAACCAGACCGAAGATGGTGGTCTGCCGCAGTCCGGTCGCATCGTTGTAGATTATATGGTGCAGAAAAGCGGTAAAACCGGTACCATCACCTACCAGCGTGGCATCCTGCTGCCGCAGAAAGTTTGGTGCGCTTCCGGTCGTAGCAAAGTAATCAAAGGTTGATAG FOR PRIMER SEQ ID No: 193CGGATAACAATTCCCCTCTAG REV PRIMER SEQ ID No: 194CGAAGTGAGATTTGGTTGGAGTAGTGGTCGCTAACAGAGACAGCACGTTC FOR PRIMERSEQ ID No: 195 CCCGCAGAACGTGCTGTCTCTGTTAGCGACCACTACCCCAACCAAATCTCREV PRIMER SEQ ID No: 196 CTATTTCACCCAAATTGGAC REV PRIMER SEQ ID No: 197CACAGCTTGCCACGGGTTTCAGTGCCTTTCGCTAACAGAGACAGCACGTTC FOR PRIMERSEQ ID No: 198 CCCGCAGAACGTGCTGTCTCTGTTAGCGAAAGGCACTGAAACCCGTGGCREV PRIMER SEQ ID No: 199 GACTAGACGCTCAGCTATCAACCTTTGATTACTTTGCTACGACCREV PRIMER SEQ ID No: 200CACAGCTTACCGCGGGTACGAGTGCCTTTCGCTAACAGAGACAGCACGTTC FOR PRIMERSEQ ID No: 201 CGGGATCCAAAGGCACTCGTACCCGCGGTAAG REV PRIMERSEQ ID No: 202 GACTAGACGCTCAGCTATCAGCCCTTAATGACCTTGGAACGGCC REV PRIMERSEQ ID No: 203 GCATAGTTTTCCTCTGGTCTGTGTTCCTTTCGCTAACAGAGACAGCACGTTCFOR PRIMER SEQ ID No: 204 AAAGGAACACAGACCAGAGG REV PRIMER SEQ ID No: 205CTACTACCCTTTTATTACCTTGCTCC REV PRIMER SEQ ID No: 206CACAGCTTGCCACGGGTTTTAGTGCCTTTCGCTAACAGAGACAGCACGTTC FOR PRIMERSEQ ID No: 207 AAAGGCACTAAAACCCGTGGC REV PRIMER SEQ ID No: 208GACTAGACGCTCAGCTATCAACCTTTGATTACTTTGCTACGACC STF2.HA1-1(MAL)SEQ ID NO: 209MAQVINTNSLSLLTQNNLNKSQSALGTAIERLSSGLRINSAKDDAAGQAIANRFTANIKGLTQASRNANDGISIAQTTEGALNEINNNLQRVRELAVQSANSTNSQSDLDSIQAEITQRLNEIDRVSGQTQFNGVKVLAQDNTLTIQVGANDGETIDIDLKQINSQTLGLDSLNVQKAYDVKDTAVTTKAYANNGTTLDVSGLDDAAIKAATGGTNGTASVTGGAVKFDADNNKYFVTIGGFTGADAAKNGDYEVNVATDGTVTLAAGATKTTMPAGATTKTEVQELKDTPAVVSADAKNALIAGGVDATDANGAELVKMSYTDKNGKTIEGGYALKAGDKYYAADYDEATGAIKAKTTSYTAADGTTKTAANQLGGVDGKTEVVTIDGKTYNASKAAGHDFKAQPELAEAAAKTTENPLQKIDAALAQVDALRSDLGAVQNRFNSAITNLGNTVNNLSEARSRIEDSDYATEVSNMSRAQILQQAGTSVLAQANQVPQNVLSLLATTTPTKSHFANLKGTETRGKLCPKCLNCTDLDVALGRPKCTGNIPSARVSILHEVRPVTSGCFPIMHDRTKIRQLPNLLRGYEHIRLSTHNVINAENAPGGSYKIGTSGSCPNVTNGNGFFATMAWAVPKNDNNKTATNSLTIEVPYICTEGEDQITVWGFHSDNEAQMAKLYGDSKPQKFTSSANGVTTHYVSQIGGFPNQTEDGGLPQSGRIVVDYMVQKSGKTGTITYQRGILLPQKVWCASGRSKVIKGSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVK STF2.HA1-2(MAL) SEQ ID NO: 210MAQVINTNSLSLLTQNNLNKSQSALGTAIERLSSGLRINSAKDDAAGQAIANRFTANIKGLTQASRNANDGISIAQTTEGALNEINNNLQRVRELAVQSANSTNSQSDLDSIQAEITQRLNEIDRVSGQTQFNGVKVLAQDNTLTIQVGANDGETIDIDLKQINSQTLGLDSLNVQKAYDVKDTAVTTKAYANNGTTLDVSGLDDAAIKAATGGTNGTASVTGGAVKFDADNNKYFVTIGGFTGADAAKNGDYEVNVATDGTVTLAAGATKTTMPAGATTKTEVQELKDTPAVVSADAKNALIAGGVDATDANGAELVKMSYTDKNGKTIEGGYALKAGDKYYAADYDEATGAIKAKTTSYTAADGTTKTAANQLGGVDGKTEVVTIDGKTYNASKAAGHDFKAQPELAEAAAKTTENPLQKIDAALAQVDALRSDLGAVQNRFNSAITNLGNTVNNLSEARSRIEDSDYATEVSNMSRAQILQQAGTSVLAQANQVPQNVLSLLAKGTETRGKLCPKCLNCTDLDVALGRPKCTGNIPSARVSILHEVRPVTSGCFPIMHDRTKIRQLPNLLRGYEHIRLSTHNVINAENAPGGSYKIGTSGSCPNVTNGNGFFATMAWAVPKNDNNKTATNSLTIEVPYICTEGEDQITVWGFHSDNEAQMAKLYGDSKPQKFTSSANGVTTHYVSQIGGFPNQTEDGGLPQSGRIVVDYMVQKSGKTGTITYQRGILLPQKVWCASGRSKVIKG STF2.HA1-2(SH) SEQ ID NO: 211MAQVINTNSLSLLTQNNLNKSQSALGTAIERLSSGLRINSAKDDAAGQAIANRFTANIKGLTQASRNANDGISIAQTTEGALNEINNNLQRVRELAVQSANSTNSQSDLDSIQAEITQRLNEIDRVSGQTQFNGVKVLAQDNTLTIQVGANDGETIDIDLKQINSQTLGLDSLNVQKAYDVKDTAVTTKAYANNGTTLDVSGLDDAAIKAATGGTNGTASVTGGAVKFDADNNKYFVTIGGFTGADAAKNGDYEVNVATDGTVTLAAGATKTTMPAGATTKTEVQELKDTPAVVSADAKNALIAGGVDATDANGAELVKMSYTDKNGKTIEGGYALKAGDKYYAADYDEATGAIKAKTTSYTAADGTTKTAANQLGGVDGKTEVVTIDGKTYNASKAAGHDFKAQPELAEAAAKTTENPLQKIDAALAQVDALRSDLGAVQNRFNSAITNLGNTVNNLSEARSRIEDSDYATEVSNMSRAQILQQAGTSVLAQANQVPQNVLSLLAKGTRTRGKLCPDCLNCTDLDVALGRPMCVGTTPSAKASILHEVRPVTSGCFPIMHDRTKIRQLPNLLRGYENIRLSTQNVIDAEKALGGPYRLGTSGSCPNATSKSGFFATMAWAVPKDNNKNATNPLTVEVPYICTEGEDQITVWGFHSDDKTQMKNLYGDSNPQKFTSSANGVTTHYVSQIGGFPDQTEDGGLPQSGRIVVDYMVQKPGKTGTIVYQRGVLLPQKVWCASGRSKVIKG STF2 SEQ ID NO: 212ATGGCACAAGTAATCAACACTAACAGTCTGTCGCTGCTGACCCAGAATAACCTGAACAAATCCCAGTCCGCACTGGGCACCGCTATCGAGCGTCTGTCTTCTGGTCTGCGTATCAACAGCGCGAAAGACGATGCGGCAGGTCAGGCGATTGCTAACCGTTTCACCGCGAACATCAAAGGTCTGACTCAGGCTTCCCGTAACGCTAACGACGGTATCTCCATTGCGCAGACCACTGAAGGCGCGCTGAACGAAATCAACAACAACCTGCAGCGTGTGCGTGAACTGGCGGTTCAGTCTGCTAACAGCACCAACTCCCAGTCTGACCTCGACTCCATCCAGGCTGAAATCACCCAGCGCCTGAACGAAATCGACCGTGTATCCGGCCAGACTCAGTTCAACGGCGTGAAAGTCCTGGCGCAGGACAACACCCTGACCATCCAGGTTGGCGCCAACGACGGTGAAACTATCGATATCGATCTGAAGCAGATCAACTCTCAGACCCTGGGTCTGGACTCACTGAACGTGCAGAAAGCGTATGATGTGAAAGATACAGCAGTAACAACGAAAGCTTATGCCAATAATGGTACTACACTGGATGTATCGGGTCTTGATGATGCAGCTATTAAAGCGGCTACGGGTGGTACGAATGGTACGGCTTCTGTAACCGGTGGTGCGGTTAAATTTGACGCAGATAATAACAAGTACTTTGTTACTATTGGTGGCTTTACTGGTGCTGATGCCGCCAAAAATGGCGATTATGAAGTTAACGTTGCTACTGACGGTACAGTAACCCTTGCGGCTGGCGCAACTAAAACCACAATGCCTGCTGGTGCGACAACTAAAACAGAAGTACAGGAGTTAAAAGATACACCGGCAGTTGTTTCAGCAGATGCTAAAAATGCCTTAATTGCTGGCGGCGTTGACGCTACCGATGCTAATGGCGCTGAGTTGGTCAAAATGTCTTATACCGATAAAAATGGTAAGACAATTGAAGGCGGTTATGCGCTTAAAGCTGGCGATAAGTATTACGCCGCAGATTACGATGAAGCGACAGGAGCAATTAAAGCTAAAACTACAAGTTATACTGCTGCTGACGGCACTACCAAAACAGCGGCTAACCAACTGGGTGGCGTAGACGGTAAAACCGAAGTCGTTACTATCGACGGTAAAACCTACAATGCCAGCAAAGCCGCTGGTCATGATTTCAAAGCACAACCAGAGCTGGCGGAAGCAGCCGCTAAAACCACCGAAAACCCGCTGCAGAAAATTGATGCCGCGCTGGCGCAGGTGGATGCGCTGCGCTCTGATCTGGGTGCGGTACAAAACCGTTTCAACTCTGCTATCACCAACCTGGGCAATACCGTAAACAATCTGTCTGAAGCGCGTAGCCGTATCGAAGATTCCGACTACGCGACCGAAGTTTCCAACATGTCTCGCGCGCAGATTCTGCAGCAGGCCGGTACTTCCGTTCTGGCGCAGGCTAACCAGGTCCCGCAGAACGTGCTGTCTCTGTTACGT B/Yamagata/16/18 HA SEQ ID NO: 213MKAIIVLLMVVTSNADRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTPTKSHFANLKGTKTRGKLCPNCLNCTDLDVALGRPMCMGTIPSAKASILHEVRPVTSGCFPIMHDRTKIRQLPNLLRGYENIRLSTHNVINAERAPGGPYRLGTSGSCPNVTSRNGFFATMAWAVPRDNKTATNPLTVEVPYICTKGEDQITVWGFHSDDKTQMKNLYGDSNPQKFTSSANGVTTHYVSQIGDFPNQTEDGGLPQSGRIVVDYMVQKPGKTGTIVYQRGVLLPQKVWCASGRSKVIKGSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLKERGFFGAIAGFLEGGWEGMIAGWHGYTSHGAHGVAVAADLKSTQEAINKITKNLNSLSELEVKNLQRLSGAMDELHNEILELDEKVDDLRADTISSQIELAVLLSNEGIINSEDEHLLALERKLKKMLGPSAVDIGNGCFETKHKCNQTCLDRIAAGTFNAGEFSLPTFDSLNITAASLNDDGLDNHTILLYYSTAASSLAVTLMIAIFIVYMVSRDNVSCSICL B/Victoria/2/87 HASEQ ID NO: 787MKAIIVLLMVVTSNADRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTPTKSHFANLKGTKTRGKLCPKCLNCTDLDVALGRPKCTGTIPSAKASILHEVKPVTSGCFPIMHDRTKIRQLPNLLRGYEHIRLSTHNVINAETAPGGPYKVGTSGSCPNVTNGNGFFATMAWAVPKNDNNKTATNPLTVEVPYICTEGEDQITVWGFHSDNEAQMVKLYGDSKPQKFTSSANGVTTHYVSQIGGFPNQAEDGGLPQSGRIVVDYMVQKSGKTGTITYQRGILLPQKVWCASGRSKVIKGSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLKEKGFFGAIAGFLEGGWEGMIAGWHGYTSHGAHGVAVAADLKSTQEAINKITKNLNSLSELEVKNLQRLSGAMDELHNKILELDEKVDDLRADTISSQIELAVLLSNEGIINSEDEHLLALERKLKKMLGPSAVEIGNGCFETKHKCNQTCLDRIAAGTFNAGEFSLPTFDSLNITAASLNDDGLDNHTILLYYSTAASSLAVTLMIAIFIVYMVSRDNVSCSICL

Example 13 Flagellin Proteins with Engineered Cysteine Residues forChemical Conjugation

The crystal structure for the bacterial flagellin S. typhimuriumflagellin type 2 (STF2, SEQ ID NO: 312) has been reported (Yonekura, K.,et al., Nature 424:643-650 (2003)). Complete atomic model of thebacterial flagellar filament by electron cryomicroscopy has beenreported, and the most detailed structure-function study of the TLR5activation was based on this structure (Smith, K. D., et al., NatureImmunology 4:1247-1253 (2003)). Toll-like Receptor 5 (TLR5) recognizes aconserved site on flagellin required for protofilament formation andbacterial motility (Nature Immunology 4(12):1247-1253). Mutationalanalysis demonstrated that the TLR5 activity of flagellin resides in tworegions in the N- and C-terminal domains, stretches of 39 and 31 aminoacids, respectively (Smith, et al.). These regions are highlighted ingrey in FIG. 18. Alanine-scanning mutagenesis of these regionsidentified a number of mutations which reduced TLR5 activation, but nosingle mutation completely abrogated activity, suggesting that there isa degree of flexibility or redundancy in the TLR5 binding site. Thismakes sense from an evolutionary perspective, otherwise the bacteriacould easily evolve to evade detection by TLR5. Deletion of thehypervariable hinge region and retention of the conserved N- andC-terminal domains results in a protein (STF2Δ) that retains full TLR5activity, demonstrating that the N- and C-terminal domains (together)are necessary and sufficient for TLR5 activation.

Lysine residues are convenient substrates for chemical conjugation ofdiverse chemical structures to a protein carrier such as flagellin. Ofthe 30 lysine residues in STF2 (SEQ ID NO: 312), only one lies withinthe experimentally-defined TLR5 activation site, and this particularlysine is not conserved among all bacterial flagellins (Smith, et al.,Nature Immunology 4:1247-1253 (2003)). Of the remaining 29 lysines, 24are located in the hypervariable hinge region of flagellin, thus leavingonly 5 lysine residues in STF2Δ, (SEQ ID NO: 313). The structure inFIGS. 19 and 20 shows that most lysines are spatially distal to the TLR5activation domain, thus it would seem that lysines could be randomlyconjugated with peptide or carbohydrate antigens with a reasonableprobability of not interfering with the TLR5 activity of the resultingconjugated STF2Δ, (SEQ ID NO: 313) protein. Five lysines appear toborder the TLR5 activation site quite closely; these residues would bethe first to consider mutating if antigen conjugation to lysine affectsTLR5 activity.

Possible additional sites for conjugation (such as cysteines oradditional lysines) may be engineered into full-length flagellin (STF2(SEQ ID NO: 312)) or STF2Δ (SEQ ID NO: 313) by placing such residues inthe hypervariable region or in the tail of the conserved N- andC-terminal domains distal to the TLR5 binding site. Chemical methods forconjugation to other amino acids also exist. These include carboxy aminoacids (glutamic acid, aspartic acid) and the carboxyl terminal aminoacid, arginine, histidine, tryptophan, tyrosine, and serine. Any ofthese strategies may be utilized to conjugate antigenic structures toflagellin without interfering with the TLR5 activation site.

Some types of antigens including polysaccharides are not amenable torecombinant fusion DNA technology nor to synthetic peptide chemistry,and thus cannot be genetically or synthetically linked to a ligand for aToll-like Receptor (TLR). In addition, it is possible that genetic orsynthetic fusion of a peptide to a TLR ligand may inhibit the properfolding of the TLR ligand. Chemical conjugation of the antigen to theTLR ligand, which is folded and purified, may be useful. The chemicalconjugation of peptide antigens also permits the placement of antigen atspecific sites on the TLR ligand, thus limiting interference withreceptor binding of the TLR ligand or maximizing exposure to antigenreceptors. The conjugation of many peptide antigens to a TLR ligand mayalso maximize immunogenicity by increasing the conformationalheterogeneity with which antigen is presented to the immune system. Toprovide a site for chemical conjugation of such antigens to flagellin, acysteine residue was engineered at different sites in the gene encodingSTF2Δ (flagellin with the hypervariable hinge region deleted; SEQ ID NO:313 and SEQ ID NO: 314).

Materials and Methods

Polymerase Chain Reaction (PCR):

Platinum® PCR SuperMix High Fidelity kit (catalog number 12532-016,Invitrogen Corporation, Carlsbad, Calif.) was used for all PCRamplifications using the following protocol based on the manufacturer'sinstructions.

-   -   1. The following components were added in any order to each        reaction tube:        -   a. 45 μl Platinum® PCR SuperMix High Fidelity (PCR reaction            buffer)        -   b. Primer solution (10 pMol final concentration of each            primer)        -   c. Template DNA solution (10 ng plasmid DNA)    -   2. Reaction volume was made up to 50 μl with water    -   3. Tubes were capped and loaded in thermal cycler    -   4. Tubes were incubated at 94° C. for 30 to 120 seconds to        completely denature the template and activate the enzyme    -   5. The following PCR amplification was performed for 25-35        cycles:        -   a. Denature at 94° C. for 15-30 seconds        -   b. Anneal at 50-55° C. for 15-30 seconds        -   c. Extend at 68-72° C. for 1 minute per kb of PCR product            size        -   d. If necessary, cool to 4° C. and hold until ready for next            process

Construction of STF2Δ Gene (SEQ ID NO: 314):

Full length flagellin of Salmonella typhimurium fljb (STF2, SEQ ID NO:312) is encoded by a 1.5 kb gene (SEQ ID NO: 315). To generate STF2Δ(SEQ ID NO: 314), the sequence corresponding to the hypervariable hingeregion (amino acids 170 to 415 of SEQ ID NO: 312) was deleted andreplaced with a short flexible linker (GAPVDPASPW (SEQ ID NO: 336))designed to facilitate interactions of the NH2 and COOH terminalsequences of STF2 (SEQ ID NO: 315) necessary for TLR5 signaling (Smith,K. D., et al., (2004). Toll-like Receptor 5 recognizes a conserved siteon flagellin required for protofilament formation and bacterial motility(Smith, et al., Nature Immunology 4:1247-53 (2003)).

To generate the STF2Δ plasmid, a two-step PCR was used (FIG. 21). In thefirst reaction, a plasmid encoding STF2.OVA (a fusion of full-lengthSTF2 with full-length ovalbumin, SEQ ID NO: 316) was mixed with primersSTF28BGF-1 (SEQ ID NO: 317) and STF28MCR-1 (SEQ ID NO: 318) in PCRreaction buffer, and the mixture was amplified in a PCR reaction asdescribed above. In a parallel reaction, STF2.OVA template plasmid wasmixed with primers STF28.MCF-2 (SEQ ID NO: 319) and STF28ECR-2 (SEQ IDNO: 320) in PCR reaction buffer, and the mixture was amplified in a PCRreaction as described above.

The PCR amplification reactions generated ˜500 bp and 270 bp fragments,respectively. These PCR products were combined with primers STF28BGF-1(SEQ ID NO: 317) and STF28ECR-2 (SEQ ID NO: 320) in PCR reaction buffer,and the mixture was amplified in a PCR reaction as described above. Theamplified DNA product from this reaction (770 bp) was digested withBglII and EcoRI restriction enzymes for 2 hours at 37° C., and ligatedinto pMTBiP/V5-His B (Invitrogen, Carlsbad, Calif.) that had previouslybeen digested with BglII and EcoRI and treated with calf intestinalphosphatase (CIP). An aliquot of the ligation mix was used to transformE. coli TOP10 cells. The resultant construct pMT/STF2Δ was used togenerate the cysteine modified STF2Δ constructs.

Construction of STF2Δ Genes Engineered to Express a Single CysteineResidue:

To introduce cysteine residues at defined positions in the STF2polypeptide, pMT/STF2Δ was used as a template in PCR with primer pairsas defined below.

To construct the STF2Δ.3′Cys (SEQ ID NO: 325) plasmid, pMT/STF24 plasmidwas mixed with 3′Forward1 (SEQ ID NO: 321) and 3′Reverse1 (SEQ ID NO:322) primers in PCR reaction buffer, and the mixture was amplified in aPCR reaction as described above.

To construct the 5′Cys.STF2Δ (SEQ ID NO: 326) plasmid, pMT/STF24 plasmidwas mixed with 5′Forward2 (SEQ ID NO: 323) and 5′Reverse2 (SEQ ID NO:324) primers in PCR reaction buffer, and the mixture was amplified in aPCR reaction as described above.

For STF2Δ.3′Cys (SEQ ID NO: 325) and 5′Cys.STF2Δ (SEQ ID NO: 326)constructs, the PCR products generated were digested with Nde1 and Blp1restriction enzymes and purified by agarose gel electrophoresis. Thepurified fragments were ligated by compatible ends to pET24a (Novagen,Madison, Wis.) plasmid DNA that had been previously digested with Nde1and Blp1 restriction enzymes and CIP-treated. The resulting constructsare designated pET/STF2Δ.3′Cys and pET/5′Cys.STF2Δ, respectively.

To construct the STF2Δ.HingeCys (SEQ ID NO: 331) plasmid, a two step PCRsimilar to that described in FIG. 18 was employed. One fragment wasgenerated by mixing pMT/STF2Δ with paired HingeForward1 (SEQ ID NO: 327)and HingeReverse3 (SEQ ID NO: 328) primers in PCR reaction buffer, andthe mixture was amplified in a PCR reaction as described above. Anotherfragment was generated by mixing pMT/STF2Δ with paired HingeForward2(SEQ ID NO: 329) and HingeReverse4 (SEQ ID NO: 330) primers in PCRreaction buffer, and the mixture was amplified in a PCR reaction asdescribed above. Aliquots from each of these PCR reactions were combinedand mixed with HingeForward1 (SEQ ID NO: 327) and HingeReverse4 (SEQ IDNO: 330) primers in PCR reaction buffer, and the mixture was amplifiedin a PCR reaction as described above. The final PCR product was purifiedby agarose gel electrophoresis and digested with Nde1 and Blp1, and thepurified fragment was ligated into the pET24a vector as described above.The resulting construct is designated pET/STF2Δ.HingeCys.

Protein Expression:

Plasmids pET/STF2Δ.3′Cys, pET/5′Cys.STF2Δ, and pET/STF2Δ.HingeCys weretransformed into competent E. coli BLR(DE3) cells as follows. To a1.5-ml snap-cap polypropylene tubes pre-chilled on ice was added 20 μlaliquot of BLR (DE3) cells and 1 μl plasmid DNA (1 μg/μl), and themixture was incubated for 30 minutes. The tubes were heated for exactly30 seconds in a 42° C. water bath without shaking, and then placed onice for an additional 2 minutes. To each tube of heat shocked cells wereadded 250 μl of room temperature SOC medium.

The cells were recovered at 37° C. (shaking at 250 rpm) for 60 minutesprior to plating on selective media containing kanamycin. Variousaliquots (50-100 μl) of the transformation mix were plated and theplates were incubated at 37° C. for 15 to 18 hours. Colonies were pickedand inoculated into 2 ml Luria-Bertani (LB) broth supplemented with 25μg/ml kanamycin and 12.5 μg/ml tetracycline, and cultured overnight.Fresh LB cultures were inoculated by diluting an aliquot of theovernight cultures 1:100 and cultured at 37° C. with shaking. When theOD₆₀₀ of the culture reached 0.6 to 1.0, protein expression was inducedby the addition of isopropyl thio-β-D-galactoside (IPTG) to a finalconcentration of 1 mM. Several hours after induction, the cells wereharvested for analysis of protein expression by SDS-PAGE. Glycerolstocks were prepared from cultures grown in LB supplemented with 0.5%glucose, 25 μg/ml kanamycin and 12.5 μg/ml tetracycline and were frozenat −80° C. following the addition of glycerol (7% final).

Protein Purification:

Proteins STF2Δ.3′Cys (SEQ ID NO: 332) and STF2Δ.HingeCys (SEQ ID NO:333) were expressed and purified as follows. Glycerol stocks of E. coliBLR(DE3) cells harboring the desired plasmids were inoculated into 10 Lshake-flasks containing LB and incubated at 37° C. with constantshaking. When cultures reached an optical density of A₆₀₀=0.8, proteinexpression was induced by the addition of 1 mM IPTG and cultures wereincubated at 37° C. with constant shaking for 4 hours before harvesting.Cells were collected from 10 L of culture by low-speed centrifugation at5,000 rpm (SLA3000 rotor) for 10 minutes and suspended in 50 mM Tris-HClpH 8.0, 1 mM EDTA, 1 mM DTT (100 ml/10 L).

The cells were disrupted by passing the cell suspension twice through amicrofluidizer at 18,000 psi. The insoluble material was separated fromsoluble proteins by centrifugation at 10,000 rpm (SS34 rotor) for 15minutes. Under the culture conditions described, all STF2Δ proteinsfractionated with the insoluble material and formed stable inclusionbodies that were collected as a solid pellet following centrifugation.The inclusion bodies were washed twice with 50 mM Tris-HCl, pH 8.0, 0.1M NaCl and 0.5% Triton X-100, followed by two washes with the samebuffer without detergent. Each wash was performed using a douncehomogenizer and the cleaned inclusion bodies (IBs) were collected bycentrifugation at 10,000 rpm (SS34 rotor) for 15 minutes. The purifiedIBs were washed a final time with 50 mM Tris-HCl, pH 8.0 and stored as acell pellet at −80° C. until needed.

IB material was thawed and solubilized in 8 M urea at pH 4.0. This stepselectively solubilized the target protein while leaving a significantamount of debris and contaminating proteins as a solid precipitate whichwas removed by centrifugation. Because of the single cysteine present inthese proteins, all subsequent purification procedures were carried outusing buffers containing 1 mM DTT as a reducing agent. Solubilizedprotein was captured using SP fast flow sepharose (30 ml, XK16) andselectively eluted with 8 M urea in 25 mM Na Acetate (C₂H₃O₂Na), pH 4.0,1 mM DTT, 1 mM EDTA and 0.2 M NaCl. To eliminate protein precipitationfollowing this step, the pH of the SP elution was adjusted from 4.0 to8.0 by dialysis against 50 mM Tris-HCl, pH 8.0, 1 mM DTT, 1 mM EDTAbefore protein refolding. The dialyzed material was refolded by directdilution (10-fold) into 50 mM Tris-HCl pH 8.0, 1 mM DTT and 1 mM EDTAsuch that the final protein concentration was less than 0.1 mg/ml. Therefolded SP pool was loaded directly onto Q high performance sepharose(30 ml, XK16) and bound protein was eluted with a 20 column volumelinear gradient from 0 to 0.5 M NaCl in 50 mM Tris-HCl, pH 8.0, 1 mMDTT, 1 mM EDTA. This chromatography step yielded a single peak thateluted at a conductivity of approximately 15 ms/cm. The eluted materialwas pooled and stored at −80° C.

Protein Characterization:

Proteins were characterized for purity, identity, endotoxin content, andbiological activity using the following assays.

SDS-PAGE: Proteins (typically 5 μg) were diluted in SDS-PAGE samplebuffer (1% SDS, 30 mM Tris-HCl, pH 6.8, 4% glycerol, 0.1 mg/mlbromophenol blue) with and without 5 mM β-mercaptoethanol. The sampleswere boiled for 5 minutes and loaded onto a 4-20% SDS polyacrylamidegel. Following electrophoresis, gels were stained with coomassie blue tovisualize protein bands.

Endotoxin assay: Endotoxin levels were measured using the QCL-1000Quantitative Chromogenic LAL test kit (BioWhittaker #50-648U), followingthe manufacturer's instructions for the microplate method.

Protein Assay: Protein concentrations were determined by the MicroBCA

Protein Assay Reagent Kit in a 96-well format using BSA as a standard(Pierce Biotechnology), following the manufacturer's instructions.

Flagellin ELISA: Protein integrity and concentration were examined byELISA with antibodies specific for flagellin. 96-well ELISA plates werecoated overnight at 4° C. with serial dilutions of each target protein,in PBS starting at 5 μg/ml. Plates were blocked with 200 μl/well ofAssay Diluent Buffer (ADB; BD Pharmingen) for one hour at roomtemperature then washed three times in phosphate-buffered salinecontaining Tween-20 (PBS-T, 12 mM NaPO₄, 137 mM NaCl, 2.7 mM KCl, 0.05%Tween 20). Rabbit polyclonal anti-flagellin antibody diluted in ADB (100μl/well, 1:5000) was added to all wells and the plates were incubatedfor 1 hour at room temperature or overnight at 4° C., then washed threetimes with PBS-T. HRP-labeled goat anti-rabbit IgG antibody (JacksonImmunochemical) diluted in ADB was added (100 μl/well, 1:5000) and theplates were incubated at room temperature for 1 hour. The plates werewashed three times with PBS-T. After adding TMB Ultra substrate (Pierce)and monitoring color development, A₄₅₀ was measured on a Tecan Farcytemicroplate spectrophotometer.

TLR5 bioactivity assay: HEK293 cells (ATCC, Cat#CRL-1573, Manassas, Va.)constitutively express TLR5, and secrete several soluble factors,including IL-8, in response to TLR5 signaling. Cells were seeded in 96well microplates (50,000 cells/well), and recombinant test proteins wereadded. The next day, the conditioned medium was harvested, transferredto a clean 96-well microplate, and frozen at −20° C. After thawing, theconditioned medium was assayed for the presence of IL-8 in a sandwichELISA using an anti-human IL-8 matched antibody pair (Pierce; Rockford,Ill., #M801E and #M802B) following the manufacturer's instructions.Optical density was measured using a microplate spectrophotometer

Results and Discussion

Protein Yield and Purity:

The final yield and endotoxin levels of each protein are shown below.

Protein SEQ ID NO: Yield (mg) Endotoxin (EU/μg) STF2Δ.3′Cys 332 75.00.01 STF2Δ.HingeCys 333 117.0 0.004

Flagellin Integrity:

Engineering a single cysteine into STF2Δ does not diminish itsrecognition by flagellin-specific antibodies, as shown in FIG. 22.

TLR5 Agonist Activity in the HEK293 IL-8 Assay:

Engineering a single cysteine into STF2Δ does not diminish the TLRagonist activity of the protein, as shown in FIG. 23. Cells exposed toSTF2Δ.3′Cys (SEQ ID NO: 332) or STF2Δ.HingeCys (SEQ ID NO: 333) secretedIL-8 at levels comparable to those induced by exposure to full-lengthflagellin (STF2, SEQ ID NO: 312) or STF2Δ without cysteine residues (SEQID NO: 313).

Fusion of full-length flagellin (STF2, SEQ ID NO: 312) or hingeregion-deleted flagellin (STF2Δ, SEQ ID NO: 313) to a protein antigensuch as West Nile virus envelope protein or influenza A hemagglutinatinsignificantly increases the immunogenicity of the fused antigen. Thisapproach is useful for protein or peptide antigens which can be encodedin a genetic fusion construct expressing both flagellin and the antigenof interest in a single protein, but it cannot be applied to non-proteinor non-peptide antigens, such as polysaccharide antigens. Since manybacteria and tumor cells express specific polysaccharide structures thatare antigenic but poorly immunogenic, it is important to devise scalablemethods for increasing the immunogenicity and protective efficacy ofsuch structures. Chemical conjugation to a TLR ligand, such asflagellin, is one potential strategy.

There are several methods of chemical linkage, but one of the simplestis to couple an antigen to a free thiol group such as that on a single,unmatched cysteine residue. Since flagellin does not natively containany cysteines, cysteine residues were engineered into flagellin at oneof three positions: the amino terminus of flagellin (5′Cys.STF2Δ, SEQ IDNO: 326 and SEQ ID NO: 334), the carboxy terminus of flagellin(STF2Δ.3′Cys, SEQ ID NO: 325 and SEQ ID NO: 332), and in the deletedhinge region of flagellin (STF2Δ.HingeCys, SEQ ID NO: 331 and SEQ ID NO:333). All constructs were produced in the expression vector pET24a forE. coli expression. When purified from E. coli cells, STF2Δ.HingeCys(SEQ ID NO: 333) and STF2Δ.3′Cys (SEQ ID NO: 332) proteins retain allantigenic and TLR5 biological activity properties of the unmodifiedSTF2Δ protein, thus confirming that the introduction of a singlecysteine residue does not negatively impact the expression,purification, refolding, or biological activity of STF2Δ.

Conjugation of Influenza Hemagglutinin Maturational Cleavage SitePeptide to STF2Δ.hingeCYS

A peptide representing the maturational cleavage site of influenzahemagglutinin was chemically conjugated a modified flagellin protein,STF2Δ.HingeCys (SEQ ID NO: 333).

Materials and Methods

Production of STF2Δ.HingeCys Protein:

STF2Δ.HingeCys protein (SEQ ID NO: 333) was expressed and purified asdescribed in the previous Example.

Synthesis of H1C1 Peptides

The sequence NH₂-NIPSIQSRGLFFAIAGFIE-COOH (SEQ ID NO: 337) representsthe maturational cleavage site of influenza A/H1N1 hemagglutinin(Bianchi, E., et al. (2005). Universal influenza B vaccine based on thematurational cleavage site of the hemagglutinin precursor (Bianchi, etal., J. Virol. 79:7380-7388 (2005)). Two peptides were designed with anextra cysteine or an extra lysine on the N-terminus to facilitatechemical linkage to carrier proteins, resulting in the following peptidesequences:

CysH1C1 (SEQ ID NO: 338) (SEQ ID NO: 338) NH₂-CNIPSIQSRGLFFAIAGFIE-COOHLysH1C1 (SEQ ID NO: 339) (SEQ ID NO: 339) NH₂-KNIPSIQSRGLFFAIAGFIE-COOH

The peptides were synthesized by Anaspec, Inc., (San Jose, Calif.)utilizing standard Fmoc chemistry, after which they were cleaved fromthe matrix with trifluoroacetic acid (TFA), purified by reversed-phaseHPLC and lyophilized.

Conjugation of CysH1C1 Peptide to STF2Δ.HingeCys:

STF2Δ.HingeCys protein (SEQ ID NO: 333) at a concentration of 4.9 mg/mLwas dialyzed overnight into Buffer A [1× phosphate-buffered saline(PBS), 5 mM EDTA, pH 7.2]. BM(PEO)₂ (Pierce Biotechnology, Rockford,Ill.), a homobifunctional maleimide crosslinker, was dissolved in DMSO(dimethyl sulfoxide) and added to a final concentration of 2.3 mM. Afterincubating for 1 hour at room temperature, the free crosslinker wasremoved from the protein using a 5 ml Superdex 25 HiTrap desaltingcolumn. CysH1C1 peptide (SEQ ID NO: 338) was dissolved in DMSO and addedto the maleimide-derivatized STF2Δ.HingeCys protein at a finalconcentration of 0.32 mM. After incubating 3 hours at room temperature,the reaction was stopped by adding DTT (dithiothreitol) to a finalconcentration of 40 mM. The protein and protein-peptide conjugate wereseparated from free peptide by fractionation on a Superdex 200 10/300size-exclusion chromatography (SEC) column (GE/Amersham; Piscataway,N.J.) equilibrated in 1× Tris-buffered saline (TBS), pH 8.0.

Protein Characterization:

Proteins were characterized for purity, identity, endotoxin content, andbiological activity as described in the previous Example.

Results and Discussion

Characterization of the STF2Δ.HingeCys:CysH1C1 Peptide Conjugate:

Conjugation of the CysH1C1 peptide (SEQ ID NO: 338) to STF2Δ.HingeCys(SEQ ID NO: 333) was assayed by SDS-PAGE with coomassie staining Peptideconjugation to the protein resulted in a doublet band in which thefaster-migrating band corresponded to the un-conjugated protein and theslower migrating band corresponded to the protein-peptide conjugate.

A cloudy precipitate was seen in the conjugation mixture due to the lowsolubility of the peptide. To ascertain if the protein-peptide conjugatewas precipitating, the mixture was centrifuged (16,000×g for 15 minutes)and the resulting pellet and supernatant fractions analyzed by SDS-PAGE.The majority of the peptide-conjugated protein remains in thesupernatant indicating that it is still soluble.

Size-exclusion chromatography (SEC) of the STF2Δ.HingeCys:CysH1C1conjugate was performed to separate the conjugated protein fromremaining crosslinker and unconjugated peptide. A single major proteinpeak eluted in the included range of the column at the expected volumefor monomeric STF2Δ, while there is very little material eluting in thevoid (FIG. 24), indicating that most of the fractionating protein ismonomeric.

The monomeric nature of the STF2Δ.HingeCys:CysH1C1 conjugate wasconfirmed by SDS-PAGE analysis of the S200 fractions (FIG. 25). Theprotein-peptide conjugate co-elutes with the unconjugated protein in thesame ratio as in the load sample, indicating that the conjugate speciesis a monomer.

Bioactivity of the STF2Δ.HingeCys:CysH1C1 conjugate mixture was found tobe equivalent to the unconjugated STF2Δ.HingeCys protein, indicatingthat the attachment of the BM(PEO)₂ crosslinker and the peptideconjugation reaction does not inhibit TLR5 stimulatory activity (FIG.8).

Conjugation of a PAM₃CYS-Containing Lipopeptide to an InfluenzaHemagglutinin Antigen

Several types of Toll-like Receptors (TLRs) are potently activated bylipids or lipid-conjugates. These include TLR2/1 (diacyl lipoproteinsand GPI-linked proteins), TLR2/6 (triacyl lipoproteins and GPI-linkedproteins) and TLR4 (lipopolysaccharides). The utility of vaccinescomposed of antigens liked to such lipids is obvious: the lipids arestrong activators of innate immune pathways and yet are themselvespoorly immunogenic. However, the production of such conjugates is verycomplex. Biosynthesis of recombinant lipoproteins in bacteria is limitedby low productivity and extreme difficulty in purifying the resultingprotein-lipopeptide fusion. Chemical synthesis of lipopeptide antigensis more straight forward, but is limited to peptide antigens. Here wedemonstrate the chemical conjugation of a Pam₃Cys lipopeptide, a TLR2/6agonist, to influenza HA1-1His₆ in a process which bypasses thedifficulties inherent in both biosynthesis and complete chemicalsynthesis of lipidated antigens.

Materials and Methods

Production of HA1-1His₆(PR8)Bv (SEQ ID NO: 179)

HA1-1His₆ (PR8)Bv was prepared as described herein.

Synthesis of Pam₃CS(K)₄GC (SEQ ID NO: 335)

Custom lipopeptide synthesis was performed by Anaspec, Inc. (San Jose,Calif.) using standard solid-phase Fmoc chemistry, after which thelipopeptide was cleaved from the matrix with trifluoroacetic acid (TFA),purified by reversed-phase HPLC and lyophilized.

Conjugation of Pam₃CS(K)₄GC (SEQ ID NO:335) to HA1-1His₆(PR8)Bv (SEQ IDNO: 179)

HA1-1His₆(PR8)Bv (SEQ ID NO: 179) was dialyzed into Buffer A (1×PBS+5 mMEDTA, pH 7.2). Sulfo-SMCC (Pierce; Rockland, Ill.) a heterobifunctionalmaleimide/NHS-ester crosslinker was dissolved in DMSO and added to afinal concentration of 0.432 mM. After incubating for 30 min. at roomtemperature the protein was desalted into Buffer A using a G-25 HiTrapdesalting column (GE/Amersham; Piscataway, N.J.). Triton X-114 (TX-114)(Sigma; St Louis, Mo.) was added to a final concentration of 1% (w/v).Pam₃CS(K)₄GC was dissolved to 20 mg/ml in DMSO and added to a finalconcentration of 0.6 mg/ml. After incubating at room temperature for 3hours, the reaction tube was placed in a 37° C. bath for 10 minutes tocause TX-114 droplet formation. The sample was then centrifuged at16,000×g for 10 minutes to separate the detergent and aqueous phases.After drawing off the aqueous phase the detergent phase was resuspendedto the original reaction volume with Buffer A. The total, detergent, andaqueous samples were then analyzed by SDS-PAGE.

Protein Characterization:

Proteins-lipopeptide conjugates were characterized by SDS-PAGE.Samples(typically 5 μg) were diluted in SDS-PAGE sample buffer (0.1MTris, pH 8.0/4% SDS/25% glycerol/0.1M DTT). The samples were boiled for5 minutes and loaded onto a 10% SDS polyacrylamide gel. Followingelectrophoresis, gels were stained with coomassie blue to visualizeprotein bands.

Results and Discussion

Input HA1-1His₆(PR8)Bv (SEQ ID NO: 179) runs as a doublet band onSDS-PAGE. This has been seen for all of the HA1-1His₆ proteins made inBaculovirus to date and is likely due to differences in glycosylation.HA1-1His₆(PR8)Bv (SEQ ID NO: 179) which has been derivatized withSulfo-SMCC but not conjugated to lipopeptide stays in the aqueous phaseduring TX-114 separation, the expected behavior for a soluble, globularprotein. HA1-1His₆(PR8)Bv (SEQ ID NO: 179) conjugated to Pam₃Cys shows 3or 4 bands of higher molecular weight than the derivatized inputprotein, consistent with covalent modification by the lipopeptide. OnTX-114 phase separation the higher mw species segregate primarily intothe detergent phase while the lower species (corresponding to theunmodified protein) stay in the aqueous phase. This behavior isconsistent with the commonly observed segregation of lipoproteins intodetergent droplets and confirms that HA1-1His₆(PR8)Bv (SEQ ID NO: 179)has been covalently modified with the Pam₃CS(K)₄GC (SEQ ID NO: 335)lipopeptide. The higher molecular-weight bands of approximately 60, 120,and 180 kDa, which are seen in the SMCC-derivatized protein but not inthe input, are covalent HA1-1 multimers formed by the crosslinker. Thismultimerization would be prevented, and the yield of singly lipidatedHA1-1 improved, by engineering a single cysteine into HA1-1 and using acysteine-specific rather than a lysine-specific crosslinker. Such astrategy would also give better control over the site of attachment ofthe lipopeptide on HA1-1, giving a more homogeneous product and possiblyimproving immunogenicity

SEQ ID NO: 312, amino acid sequence of STF2MAQVINTNSLSLLTQNNLNKSQSALGTAIERLSSGLRINSAKDDAAGQAIANRFTANIKGLTQASRNANDGISIAQTTEGALNEINNNLQRVRELAVQSANSTNSQSDLDSIQAEITQRLNEIDRVSGQTQFNGVKVLAQDNTLTIQVGANDGETIDIDLKQINSQTLGLDSLNVQKAYDVKDTAVTTKAYANNGTTLDVSGLDDAAIKAATGGTNGTASVTGGAVKFDADNNKYFVTIGGFTGADAAKNGDYEVNVATDGTVTLAAGATKTTMPAGATTKTEVQELKDTPAVVSADAKNALIAGGVDATDANGAELVKMSYTDKNGKTIEGGYALKAGDKYYAADYDEATGAIKAKTTSYTAADGTTKTAANQLGGVDGKTEVVTIDGKTYNASKAAGHDFKAQPELAEAAAKTTENPLQKIDAALAQVDALRSDLGAVQNRFNSAITNLGNTVNNLSEARSRIEDSDYATEVSNMSRAQILQQAGTSVLAQANQVPQNVLSLLR SEQ ID NO: 313, amino acid sequence of STF2ΔMAQVINTNSLSLLTQNNLNKSQSALGTAIERLSSGLRINSAKDDAAGQAIANRFTANIKGLTQASRNANDGISIAQTTEGALNEINNNLQRVRELAVQSANSTNSQSDLDSIQAEITQRLNEIDRVSGQTQFNGVKVLAQDNTLTIQVGANDGETIDIDLKQINSQTLGLDSLNVHGAPVDPASPWTENPLQKIDAALAQVDALRSDLGAVQNRFNSAITNLGNTVNNLSEARSRIEDSDYATEVSNMSRAQILQQAGTSVLAQANQVPQNVLSLLR SEQ ID NO: 314,nucleotide sequence of STF2ΔATGGCACAAGTAATCAACACTAACAGTCTGTCGCTGCTGACCCAGAATAACCTGAACAAATCCCAGTCCGCACTGGGCACCGCTATCGAGCGTCTGTCTTCTGGTCTGCGTATCAACAGCGCGAAAGACGATGCGGCAGGTCAGGCGATTGCTAACCGTTTCACCGCGAACATCAAAGGTCTGACTCAGGCTTCCCGTAACGCTAACGACGGTATCTCCATTGCGCAGACCACTGAAGGCGCGCTGAACGAAATCAACAACAACCTGCAGCGTGTGCGTGAACTGGCGGTTCAGTCTGCTAACAGCACCAACTCCCAGTCTGACCTCGACTCCATCCAGGCTGAAATCACCCAGCGCCTGAACGAAATCGACCGTGTATCCGGCCAGACTCAGTTCAACGGCGTGAAAGTCCTGGCGCAGGACAACACCCTGACCATCCAGGTTGGCGCCAACGACGGTGAAACTATCGATATCGATCTGAAGCAGATCAACTCTCAGACCCTGGGTCTGGACTCACTGAACGTGCATGGAGCGCCGGTGGATCCTGCTAGCCCATGGACCGAAAACCCGCTGCAGAAAATTGATGCCGCGCTGGCGCAGGTGGATGCGCTGCGCTCTGATCTGGGTGCGGTACAAAACCGTTTCAACTCTGCTATCACCAACCTGGGCAATACCGTAAACAATCTGTCTGAAGCGCGTAGCCGTATCGAAGATTCCGACTACGCGACCGAAGTTTCCAACATGTCTCGCGCGCAGATTTTGCAGCAGGCCGGTACTTCCGTTCTGGCGCAGGCTAACCAGGTCCCGCAGAACGTGCTGTCTCTGTTACGT SEQ ID NO: 315,nucleotide sequence of STF2ATGGACAAGTAATCAACACTAACAGTCTGTCGCTGCTGACCCAGAATAACCTGAACAAATCCCAGTCCGCACTGGGCACCGCTATCGAGCGTCTGTCTTCTGGTCTGCGTATCAACAGCGCGAAAGACGATGCGGCAGGTCAGGCGATTGCTAACCGTTTCACCGCGAACATCAAAGGTCTGACTCAGGCTTCCCGTAACGCTAACGACGGTATCTCCATTGCGCAGACCACTGAAGGCGCGCTGAACGAAATCAACAACAACCTGCAGCGTGTGCGTGAACTGGCGGTTCAGTCTGCTAACAGCACCAACTCCCAGTCTGACCTCGACTCCATCCAGGCTGAAATCACCCAGCGCCTGAACGAAATCGACCGTGTATCCGGCCAGACTCAGTTCAACGGCGTGAAAGTCCTGGCGCAGGACAACACCCTGACCATCCAGGTTGGCGCCAACGACGGTGAAACTATCGATATCGATCTGAAGCAGATCAACTCTCAGACCCTGGGTCTGGACTCACTGAACGTGCAGAAAGCGTATGATGTGAAAGATACAGCAGTAACAACGAAAGCTTATGCCAATAATGGTACTACACTGGATGTATCGGGTCTTGATGATGCAGCTATTAAAGCGGCTACGGGTGGTACGAATGGTACGGCTTCTGTAACCGGTGGTGCGGTTAAATTTGACGCAGATAATAACAAGTACTTTGTTACTATTGGTGGCTTTACTGGTGCTGATGCCGCCAAAAATGGCGATTATGAAGTTAACGTTGCTACTGACGGTACAGTAACCCTTGCGGCTGGCGCAACTAAAACCACAATGCCTGCTGGTGCGACAACTAAAACAGAAGTACAGGAGTTAAAAGATACACCGGCAGTTGTTTCAGCAGATGCTAAAAATGCCTTAATTGCTGGCGGCGTTGACGCTACCGATGCTAATGGCGCTGAGTTGGTCAAAATGTCTTATACCGATAAAAATGGTAAGACAATTGAAGGCGGTTATGCGCTTAAAGCTGGCGATAAGTATTACGCCGCAGATTACGATGAAGCGACAGGAGCAATTAAAGCTAAAACTACAAGTTATACTGCTGCTGACGGCACTACCAAAACAGCGGCTAACCAACTGGGTGGCGTAGACGGTAAAACCGAAGTCGTTACTATCGACGGTAAAACCTACAATGCCAGCAAAGCCGCTGGTCATGATTTCAAAGCACAACCAGAGCTGGCGGAAGCAGCCGCTAAAACCACCGAAAACCCGCTGCAGAAAATTGATGCCGCGCTGGCGCAGGTGGATGCGCTGCGCTCTGATCTGGGTGCGGTACAAAACCGTTTCAACTCTGCTATCACCAACCTGGGCAATACCGTAAACAATCTGTCTGAAGCGCGTAGCCGTATCGAAGATTCCGACTACGCGACCGAAGTTTCCAACATGTCTCGCGCGCAGATTCTGCAGCAGGCCGGTACTTCCGTTCTGGCGCAGGCTAACCAGGTCCCGCAGAACGTGCTGTCTCTGTTACGT SEQ ID NO: 316, nucleotide sequence of STF2.OVAATGGCACAAGTAATCAACACTAACAGTCTGTCGCTGCTGACCCAGAATAACCTGAACAAATCCCAGTCCGCACTGGGCACCGCTATCGAGCGTCTGTCTTCTGGTCTGCGTATCAACAGCGCGAAAGACGATGCGGCAGGTCAGGCGATTGCTAACCGTTTCACCGCGAACATCAAAGGTCTGACTCAGGCTTCCCGTAACGCTAACGACGGTATCTCCATTGCGCAGACCACTGAAGGCGCGCTGAACGAAATCAACAACAACCTGCAGCGTGTGCGTGAACTGGCGGTTCAGTCTGCTAACAGCACCAACTCCCAGTCTGACCTCGACTCCATCCAGGCTGAAATCACCCAGCGCCTGAACGAAATCGACCGTGTATCCGGCCAGACTCAGTTCAACGGCGTGAAAGTCCTGGCGCAGGACAACACCCTGACCATCCAGGTTGGCGCCAACGACGGTGAAACTATCGATATCGATCTGAAGCAGATCAACTCTCAGACCCTGGGTCTGGACTCACTGAACGTGCAGAAAGCGTATGATGTGAAAGATACAGCAGTAACAACGAAAGCTTATGCCAATAATGGTACTACACTGGATGTATCGGGTCTTGATGATGCAGCTATTAAAGCGGCTACGGGTGGTACGAATGGTACGGCTTCTGTAACCGGTGGTGCGGTTAAATTTGACGCAGATAATAACAAGTACTTTGTTACTATTGGTGGCTTTACTGGTGCTGATGCCGCCAAAAATGGCGATTATGAAGTTAACGTTGCTACTGACGGTACAGTAACCCTTGCGGCTGGCGCAACTAAAACCACAATGCCTGCTGGTGCGACAACTAAAACAGAAGTACAGGAGTTAAAAGATACACCGGCAGTTGTTTCAGCAGATGCTAAAAATGCCTTAATTGCTGGCGGCGTTGACGCTACCGATGCTAATGGCGCTGAGTTGGTCAAAATGTCTTATACCGATAAAAATGGTAAGACAATTGAAGGCGGTTATGCGCTTAAAGCTGGCGATAAGTATTACGCCGCAGATTACGATGAAGCGACAGGAGCAATTAAAGCTAAAACCACAAGTTATACTGCTGCTGACGGCACTACCAAAACAGCGGCTAACCAACTGGGTGGCGTAGACGGTAAAACCGAAGTCGTTACTATCGACGGTAAAACCTACAATGCCAGCAAAGCCGCTGGTCATGATTTCAAAGCACAACCAGAGCTGGCGGAAGCAGCCGCTAAAACCACCGAAAACCCGCTGCAGAAAATTGATGCCGCGCTGGCGCAGGTGGATGCGCTGCGCTCTGATCTGGGTGCGGTACAAAACCGTTTCAACTCTGCTATCACCAACCTGGGCAATACCGTAAACAATCTGTCTGAAGCGCGTAGCCGTATCGAAGATTCCGACTACGCGACCGAAGTTTCCAACATGTCTCGCGCGCAGATTTTGCAGCAGGCCGGTACTTCCGTTCTGGCGCAGGCTAACCAGGTCCCGCAGAACGTGCTGTCTCTGTTACGTCTCGAGGGCTCCATCGGCGCAGCAAGCATGGAATTTTGTTTTGATGTATTCAAGGAGCTCAAAGTCCACCATGCCAATGAGAACATCTTCTACTGCCCCATTGCCATCATGTCAGCTCTAGCCATGGTATACCTGGGTGCAAAAGACAGCACCAGGACACAAATAAATAAGGTTGTTCGCTTTGATAAACTTCCAGGATTCGGAGACAGTATTGAAGCTCAGTGTGGCACATCTGTAAACGTTCACTCTTCACTTAGAGACATCCTCAACCAAATCACCAAACCAAATGATGTTTATTCGTTCAGCCTTGCCAGTAGACTTTATGCTGAAGAGAGATACCCAATCCTGCCAGAATACTTGCAGTGTGTGAAGGAACTGTATAGAGGAGGCTTGGAACCTATCAACTTTCAACAGCTGCAGATCAAGCCAGAGAGCTCATCAATTCCTGGGTAGAAAGTCAGACAAATGGAATTATCAGAAATGTCCTTCAGCCAAGCTCCGTGGATTCTCAAACTGCAATGGTTCTGGTTAATGCCATTGTCTTCAAAGGACTGTGGGAGAAAGCATTTAAGGATGAAGACACACAAGCAATGCCTTTCAGAGTGACTGAGCAAGAAAGCAAACCTGTGCAGATGATGTACCAGATTGGTTTATTTAGAGTGGCATCAATGGCTTCTGAGAAAATGAAGATCCTGGAGCTTCCATTTGCCAGTGGGACAATGAGCATGTTGGTGCTGTTGCCTGATGAAGTCTCAGGCCTTGAGCAGCTTGAGAGTATAATCAACTTTGAAAAACTGACTGAATGGACCAGTTCTAATGTTATGGAAGAGAGGAAGATCAAAGTGTACTTACCTCGCATGAAGATGGAGGAAAAATACAACCTCACATCTGTCTTAATGGCTATGGGCATTACTGACGTGTTTAGCTCTTCAGCCAATCTGTCTGGCATCTCCTCAGCAGAGAGCCTGAAGATATCTCAAGCTGTCCATGCAGCACATGCAGAAATCAATGAAGCAGGCAGAGAGGTGGTAGGGTCAGCAGAGGCTGGAGTGGATGCTGCAAGCGTCTCTGAAGAATTTAGGGCTGACCATCCATTCCTCTTCTGTATCAAGCACATCGCAACCAACGCCGTTCTCTTCTTTGGCAGATGTGTTTCCCCTTCGAAGCTTGAAGGTAAGCCTATCCCTAACCCTCTCCTCGGTCTCGATTCTACGCGTACCGGTCATCATCACCATCACCAT TGASEQ ID NO: 317, nucleotide sequence of STF28BGF-1CTCGGGAGATCTGCACAAGTAATCAACACTAACAGTCT SEQ ID NO: 318,nucleotide sequence of STF28MCR-1CCATGGGCTAGCAGGATCCACCGGCGCTCCCTGCACGTTCA SEQ ID NO: 319,nucleotide sequence of STF28MCF-2GGAGCGCCGGTGGATCCTGCTAGCCCATGGACCGAAAACCCG SEQ ID NO: 320,nucleotide sequence of STF28ECR-2TCTGCAGAATTCACGTAACAGAGACAGCACGTTCTGCGGGACGTCCCGCAGAACGTGCTGTCTCTGTTACGTGAATTCTGCAGA SEQ ID NO: 312,nucleotide sequence of 3′Forward1 ACTGAGTGCATATGGCACAAGTAATCAACACTAACAGSEQ ID NO: 322, nucleotide sequence of 3′Reverse1GACTGACTGCTCAGCCTATTAGCAGAGCGGCCGCCACTGTGCTGGATATCTAGAG SEQ ID NO: 323nucleotide sequence of 5′Forward2AGTCAGGCCATATGTGCGCACAAGTAATCAACACTAACAGTCTG SEQ ID NO: 324,nucleotide sequence of 5′Reverse2GACTGACTGCTCAGCCTATTAACGTAACAGAGACAGCACGTTCTGCGGGACCTGGTTAGSEQ ID NO: 325, nucleotide sequence of STF2Δ.3′CysATGGACAAGTAATCAACACTAACAGTCTGTCGCTGCTGACCCAGAATAACCTGAACAAATCCCAGTCCGCACTGGGCACCGCTATCGAGCGTCTGTCTTCTGGTCTGCGTATCAACAGCGCGAAAGACGATGCGGCAGGTCAGGCGATTGCTAACCGTTTCACCGCGAACATCAAAGGTCTGACTCAGGCTTCCCGTAACGCTAACGACGGTATCTCCATTGCGCAGACCACTGAAGGCGCGCTGAACGAAATCAACAACAACCTGCAGCGTGTGCGTGAACTGGCGGTTCAGTCTGCTAACAGCACCAACTCCCAGTCTGACCTCGACTCCATCCAGGCTGAAATCACCCAGCGCCTGAACGAAATCGACCGTGTATCCGGCCAGACTCAGTTCAACGGCGTGAAAGTCCTGGCGCAGGACAACACCCTGACCATCCAGGTTGGCGCCAACGACGGTGAAACTATCGATATCGATCTGAAGCAGATCAACTCTCAGACCCTGGGTCTGGACTCACTGAACGTGCATGGAGCGCCGGTGGATCCTGCTAGCCCATGGACCGAAAACCCGCTGCAGAAAATTGATGCCGCGCTGGCGCAGGTGGATGCGCTGCGCTCTGATCTGGGTGCGGTACAAAACCGTTTCAACTCTGCTATCACCAACCTGGGCAATACCGTAAACAATCTGTCTGAAGCGCGTAGCCGTATCGAAGATTCCGACTACGCGACCGAAGTTTCCAACATGTCTCGCGCGCAGATTTTGCAGCAGGCCGGTACTTCCGTTCTGGCGCAGGCTAACCAGGTCCCGCAGAACGTGCTGTCTCTGTTACGTGAATTCTCTAGATATCCAGCACAGTGGCGGCCGCTCTGC SEQ ID NO: 326,nucleotide sequence of 5′Cys.STF2ΔATGTGCGCACAAGTAATCAACACTAACAGTCTGTCGCTGCTGACCCAGAATAACCTGAACAAATCCCAGTCCGCACTGGGCACCGCTATCGAGCGTCTGTCTTCTGGTCTGCGTATCAACAGCGCGAAAGACGATGCGGCAGGTCAGGCGATTGCTAACCGTTTCACCGCGAACATCAAAGGTCTGACTCAGGCTTCCCGTAACGCTAACGACGGTATCTCCATTGCGCAGACCACTGAAGGCGCGCTGAACGAAATCAACAACAACCTGCAGCGTGTGCGTGAACTGGCGGTTCAGTCTGCTAACAGCACCAACTCCCAGTCTGACCTCGACTCCATCCAGGCTGAAATCACCCAGCGCCTGAACGAAATCGACCGTGTATCCGGCCAGACTCAGTTCAACGGCGTGAAAGTCCTGGCGCAGGACAACACCCTGACCATCCAGGTTGGCGCCAACGACGGTGAAACTATCGATATCGATCTGAAGCAGATCAACTCTCAGACCCTGGGTCTGGACTCACTGAACGTGCATGGAGCGCCGGTGGATCCTGCTAGCCCATGGACCGAAAACCCGCTGCAGAAAATTGATGCCGCGCTGGCGCAGGTGGATGCGCTGCGCTCTGATCTGGGTGCGGTACAAAACCGTTTCAACTCTGCTATCACCAACCTGGGCAATACCGTAAACAATCTGTCTGAAGCGCGTAGCCGTATCGAAGATTCCGACTACGCGACCGAAGTTTCCAACATGTCTCGCGCGCAGATTTTGCAGCAGGCCGGTACTTCCGTTCTGGCGCAGGCTAACCAGGTCCCGCAGAACGTGCTGTCTCTGTTACGT SEQ ID NO: 327,nucleotide sequence of HingeForward1ACTGAGTGCATATGGCACAAGTAATCAACACTAACAG SEQ ID NO: 328,nucleotide sequence of HingeReverse3 GGTCCATGGGCAAGCAGGATCCACCGGCGCTSEQ ID NO: 329, nucleotide sequence of HingeFroward2AGCGCCGGTGGATCCTGCTTGCCCATGGACC SEQ ID NO: 330,nucleotide sequence of HingeReverse4GACTGACTGCTCAGCCTATTAACGTAACAGAGACAGCACGTTCTGCGGGACCTGGTTAGSEQ ID NO: 331, nucleotide sequence of STF2Δ.HingeCysATGGCACAAGTAATCAACACTAACAGTCTGTCGCTGCTGACCCAGAATAACCTGAACAAATCCCAGTCCGCACTGGGCACCGCTATCGAGCGTCTGTCTTCTGGTCTGCGTATCAACAGCGCGAAAGACGATGCGGCAGGTCAGGCGATTGCTAACCGTTTCACCGCGAACATCAAAGGTCTGACTCAGGCTTCCCGTAACGCTAACGACGGTATCTCCATTGCGCAGACCACTGAAGGCGCGCTGAACGAAATCAACAACAACCTGCAGCGTGTGCGTGAACTGGCGGTTCAGTCTGCTAACAGCACCAACTCCCAGTCTGACCTCGACTCCATCCAGGCTGAAATCACCCAGCGCCTGAACGAAATCGACCGTGTATCCGGCCAGACTCAGTTCAACGGCGTGAAAGTCCTGGCGCAGGACAACACCCTGACCATCCAGGTTGGCGCCAACGACGGTGAAACTATCGATATCGATCTGAAGCAGATCAACTCTCAGACCCTGGGTCTGGACTCACTGAACGTGCATGGAGCGCCGGTGGATCCTGCTTGCCCATGGACCGAAAACCCGCTGCAGAAAATTGATGCCGCGCTGGCGCAGGTGGATGCGCTGCGCTCTGATCTGGGTGCGGTACAAAACCGTTTCAACTCTGCTATCACCAACCTGGGCAATACCGTAAACAATCTGTCTGAAGCGCGTAGCCGTATCGAAGATTCCGACTACGCGACCGAAGTTCCAACATGTCTCGCGCGCAGATTTTGCAGCAGGCCGGTACTTCCGTTCTGGCGCAGGCTAACCAGGTCCCGCAGAACGTGCTGTCTCTGTTACGT SEQ ID NO: 332,amino acid sequence of STF2Δ.3′Cys

SEQ ID NO: 333, amino acid sequence of STF2Δ.HingeCys

SEQ ID NO: 334, amino acid sequence of 5′Cys.STF2Δ

SEQ ID NO: 335, CSKKKKGC SEQ ID NO: 336, GAPVDPASPW

Example 14

Cloning and Expression of STF2.4×H1C1, a Fusion Protein ComprisingFlagellin (TLR5Agonist) and the Maturational Cleavage Site Peptide ofInfluenza A Hemagglutinin

Materials and Methods

Construct Design:

To facilitate cloning of target genes in fusion with flagellin, acassette plasmid containing a unique BlpI site at the 3′ end of theflagellin gene was used (STF2.blp, SEQ ID NO: 340). This cassette wasmade by introducing a silent mutation (5′-GTGCTGAGCCTGTTACGT-3′) atnucleotides 1501 to 1518 of STF2, creating the unique BlpI site in theplasmid cassette, pET24/STF2.blp (SEQ ID NO: 340). Synthetic genesencoding one, two, three and four copies of the maturational cleavagesite fragment (H1C1) of influenza A subtype H1 (NIPSIQSRGLFGAIAGFIE; SEQID NO: 341) were codon-optimized for E. coli expression and obtainedfrom a commercial vendor (DNA2.0 Inc., Menlo Park, Calif.). Thesynthetic genes were excised with BlpI enzyme and ligated by compatibleends to pET24/STF2.blp which had been treated with BlpI and bacterialalkaline phosphatase (BAP). These constructs were designated asSTF2.1×H1C1, STF2.2×H1C1, STF2.3×H1C1 and STF2.4×H1C1 respectively (SEQID NO: 342; SEQ ID NO: 343; SEQ ID NO: 344; SEQ ID NO: 345).

Similarly, a fusion gene constructed to contain the H1C1 sequence andflanked by a pair of cysteines to facilitate loop formation of theconcatemers was designated as STF2.4×H1C2 (SEQ ID NO: 346). A similarapproach was employed to generate a construct harboring four tandemcopies of the cleavage fragment of influenza A subtype H5(RERRRKKRGLFGAIAGFIE; SEQ ID NO: 347) designated as constructSTF2.4×H5C1 (SEQ ID NO: 348). The consensus cleavage fragmentsrepresenting subtypes H3 (NVPEKQTRGIFGAIAGFIE; SEQ ID NO: 349), H2(NVPQIESRGLFGAIAGFIE; SEQ ID NO: 350) and the B-strain, B/HA(PAKLLKERGFFGAIAGFLE; SEQ ID NO: 351) are thus amenable to thisexperimental approach as outlined above. In each case, the constructedplasmids were used to transform competent E. coli TOP10 cells andputative recombinants were identified by PCR screening and restrictionmapping analysis.

The integrity of the constructs was verified by DNA sequencing and theywere used to transform the expression host, BLR3 (DE3) (Novagen, SanDiego, Calif.; Cat #69053). Transformants were selected on platescontaining kanamycin (50 μg/mL), tetracycline (5 μg/mL) and glucose(0.5%). Colonies were picked and inoculated into 2 ml of LB mediumsupplemented with 25 μg/ml kanamycin, 12.5 μg/ml tetracycline and 0.5%glucose and grown overnight. Aliquots of these cultures were used toinoculate fresh cultures in the same medium formulation, which werecultured until they reached an OD₆₀₀=0.6, at which time proteinexpression was induced by the addition of 1 mM IPTG and culturing for 3hours at 37° C. The cells were harvested and analyzed for proteinexpression.

SDS-PAGE and Western Blot:

Protein expression and identity were determined by gel electrophoresisand immunoblot analysis. Cells were harvested by centrifugation andlysed in Laemmli buffer. An aliquot of 10 μl of each lysate was dilutedin SDS-PAGE sample buffer with or without 100 mM DTT as a reductant. Thesamples were boiled for 5 minutes and loaded onto a 10% SDSpolyacrylamide gel and electrophoresed (SDS-PAGE). The gel was stainedwith Coomassie R-250 (Bio-Rad; Hercules, Calif.) to visualize proteinbands. For western blot, 0.5 μl/lane cell lysate was electrophoresed andelectrotransferred to a PVDF membrane and blocked with 5% (w/v) drymilk. The membrane was probed with anti-flagellin antibody 6H11 (Inotek;Beverly, Mass.) After probing with alkaline phosphatase-conjugatedsecondary antibodies (Pierce; Rockland, Ill.), protein bands werevisualized with an alkaline phosphatase chromogenic substrate (Promega,Madison, Wis.). Bacterial clones which yielded protein bands of thecorrect molecular weight and reactive with the appropriate antibodieswere selected for production of protein for use in biological assays.

Results and Discussion

Cloning and Expression of Cleavage Site Constructs:

The maturational cleavage site fragment of hemagglutinin precursor iswell-conserved across subtypes of influenza strains A and B, and couldtherefore be a target for the development of a universal vaccineeffective against most circulating strains of influenza. A series ofplasmids encoding a fusion of the TLR5 ligand with cleavage fragment wasgenerated and expressed in E. coli strain BLR(DE3). As assayed byCoomassie blue staining of the SDS-PAGE gel and confirmed by immunoblotassays, the E. coli strains harboring the constructs STF2.1×H1C1,STF2.2×H1C1, STF2.3×H1C1, and STF2.4×H1C1 displayed bands thatcorrespond to the predicted molecular weights of 55, 58, 60 and 62 kDarespectively. Similarly, BLR(DE3) strains expressing the constructsSTF2.4×H1C2 and STF2.4×H5C1 display recombinant fusion proteinsmigrating with the apparent molecular weight of 62 Kda and 66 Kdarespectively. These data indicate that a fusion of flagellin andcleavage fragment of HA is abundantly expressed in E. coli.

Expression and Purification of STF2.4×H1C1, a Fusion Protein ComprisingFlagellin (TLR5Agonist) and the Maturational Cleavage Site Peptide ofInfluenza A Hemagglutinin Materials and Methods

Bacterial Cell Growth and Cell Lysis:

The STF2.4×H1C1 (SEQ ID NO: 345) construct was expressed in the E. colihost strain BLR(DE3). The strain was retrieved from a glycerol stock andgrown in shake flasks to a final volume of 12 L. Cells were grown in LBmedium containing 50 μg/ml kanamycin, 12.5 μg/ml tetracycline, 0.5%dextrose to OD₆₀₀=0.6 and induced with 1 mM IPTG for 3 h at 37° C. Thecells were harvested by centrifugation (7000 rpm×7 minutes in a SorvallRC5C centrifuge) and resuspended in 20 mM Tris-HCl, pH 8.0, 1 μg/mlDNAseI, 1 mM PMSF, protease inhibitor cocktail and 1 mg/ml lysozyme. Thecells were then lysed by two passes through a microfluidizer(Microfluidics; Newton, Mass.) at 15,000 psi. The lysate was centrifugedat 45,000 g for one hour in a Beckman Optima L ultracentrifuge (BeckmanCoulter; Fullerton, Calif.) to separate the soluble and insolublefractions.

Purification of STF2.4×H1C1 (SEQ ID NO: 345):

After centrifugation, the supernatant fraction was collected and passedthrough a Q sepharose Fast Flow column (GE/Amersham Biosciences;Piscataway, N.J.). The flow-through fraction from this step wassupplemented with Triton X-100 (Sigma; St. Louis, Mo.) to a finalconcentration of 1% (w/v) and passed through the same Q sepharosecolumn. The flow through fraction was collected again and supplementedwith urea to a final concentration of 8 M and citric acid to a finalconcentration of 20 mM. After adjusting the pH to 3.5 with concentratedHCl, the solution was passed over a Source S column (GE/AmershamBiosciences; Piscataway, N.J.) equilibrated with 20 mM citric acid, pH3.5.

The column was then washed with 10 column volumes of equilibrationbuffer supplemented with 1% (w/v) Triton X-100 to remove endotoxin. Theprotein was then eluted in a 5-column volume linear gradient of 0 to 1 MNaCl in equilibration buffer. The STF2.4×H1C1 was then re-folded byrapid dilution to a final concentration of 0.1 mg/ml protein inrefolding buffer [0.1 M Tris-HCl, pH 8.0, 0.1 M NaC, 1% (w/v) glycerol].The refolded protein was concentrated using a pressurizedultrafiltration stir-cell (Millipore; Billerica, Mass.) and fractionatedon a Superdex 200 size-exclusion column (GE/Amersham Biosciences;Piscataway, N.J.).

SDS-PAGE and Western Blot Analysis:

Protein identity was determined, and purity estimated, by SDS-PAGE. Analiquot of 5 μg of each sample was diluted in SDS-PAGE sample bufferwith or without 100 mM DTT as a reductant. The samples were boiled for 5minutes and loaded onto a 10% SDS polyacrylamide gel (LifeGels; French'sForrest, New South Wales, AUS) and electrophoresed. The gel was stainedwith Coomassie R-250 (Bio-Rad; Hercules, Calif.) to visualize proteinbands. For western blot, 0.5 μg/lane total protein was electrophoresedas described above and then electro-transferred to a PVDF membrane andblocked with 5% (w/v) dry milk before probing with anti-flagellinantibody (Inotek; Beverly, Mass.) or serum from mice immunized with asynthetic, lipidated H1C1 peptide. After probing with alkalinephosphatase-conjugated secondary antibodies (Pierce; Rockland, Ill.),protein bands were visualized with an alkaline phosphatase chromogenicsubstrate (Promega; Madison, Wis.).

Protein Assay:

Total protein concentration for all proteins was determined using theMicro BCA (bicinchoninic acid) Assay (Pierce; Rockford Ill.) in themicroplate format, using bovine serum albumin as a standard, accordingto the manufacturer's instructions.

Endotoxin Assay:

Endotoxin levels for all proteins were determined using the QCL-1000Quantitative Chromogenic LAL test kit (Cambrex; E. Rutherford, N.J.),following the manufacturer's instructions for the microplate method.

TLR5 Bioactivity Assay:

HEK293 cells constitutively express TLR5, and secrete several solublefactors, including IL-8, in response to TLR5 signaling. Cells wereseeded in 96-well microplates (50,000 cells/well), and the STF2.4×H1C1test protein was added. The next day, the conditioned medium washarvested, transferred to a clean 96-well microplate, and frozen at −20°C. After thawing, the conditioned medium was assayed for the presence ofIL-8 in a sandwich ELISA using an anti-human IL-8 matched antibody pair(Pierce, Rockland, Ill.; #M801E and #M802B) following the manufacturer'sinstructions. Optical density was measured using a microplatespectrophotometer (FARCyte, GE/Amersham; Piscataway, N.J.).

Results and Discussion

The purified and refolded STF2.4×H1C1 protein (SEQ ID NO: 345) was foundto be substantially aggregated as the majority of the proteinfractionated in the void volume of the Superdex S200 column (FIG. 26).The protein also had poor in vitro TLR5 activity, with an EC₅₀ valueapproximately two orders of magnitude higher than the standard flagellinfusion protein STF2.OVA. The purified protein reacted with serum frommice immunized with a synthetic, lipidated H1C1 peptide.

Expression and Purification of STF2.1×H1C1, a Fusion Protein ComprisingFlagellin (TLR5Agonist) and the Maturational Cleavage Site Peptide ofInfluenza A Hemagglutinin Materials and Methods

Bacterial Cell Growth and Cell Lysis:

The STF2.1×H1C1 (SEQ ID NO: 355) construct was expressed in the E. colihost strain BLR(DE3). The strain was retrieved from a glycerol stock andgrown in shake flasks to a final volume of 12 L. Cells were grown in LBmedium containing 50 μg/ml kanamycin/12.5 μg/ml tetracycline/0.5%dextrose to OD₆₀₀=0.6 and induced with 1 mM IPTG for 3 hours at 37° C.The cells were harvested by centrifugation (7000 rpm×7 minutes in aSorvall RC5C centrifuge) and resuspended in 20 mM Tris-HCl, pH 8.0, 1μg/ml DNAseI, 1 mM PMSF, protease inhibitor cocktail and 1 mg/mllysozyme. The cells were then lysed by two passes through amicrofluidizer (Microfluidics; Newton, Mass.) at 15,000 psi. The lysatewas centrifuged at 45,000 g for one hour in a Beckman Optima Lultracentrifuge (Beckman Coulter; Fullerton, Calif.) to separate thesoluble and insoluble fractions.

Purification of STF2.1×H1C1 (SEQ ID NO: 342):

After centrifugation, the supernatant fraction was collected and passedthrough a Q sepharose Fast Flow column (GE/Amersham Biosciences;Piscataway, N.J.). The flow-through fraction from this step wassupplemented with Triton X-100 (Sigma; St. Louis, Mo.) to a finalconcentration of 1% (w/v) and passed through the same Q sepharosecolumn. The flow-through fraction was collected again and supplementedwith urea to a final concentration of 8 M and citric acid to a finalconcentration of 20 mM. After adjusting the pH to 3.5 with concentratedHCl, the solution was passed over a Source S column (GE/AmershamBiosciences; Piscataway, N.J.) equilibrated with 20 mM citric acid, pH3.5.

The column was then washed with 10 column volumes of equilibrationbuffer supplemented with 1% (w/v) Triton X-100 to remove endotoxin. Theprotein was then eluted in a 5-column volume linear gradient of 0 to 1 MNaCl in equilibration buffer. The STF2.1×H1C1 was re-folded by rapiddilution to a final concentration of 0.1 mg/ml protein in refoldingbuffer [0.1M Tris-HCl, pH 8.0/0.1M NaCl/1% (w/v) glycerol]. The refoldedprotein was concentrated using a pressurized ultrafiltration stir-cell(Millipore; Billerica, Mass.) and fractionated on a Superdex 200size-exclusion column (GE/Amersham Biosciences; Piscataway, N.J.).

SDS-PAGE and Western Blot Analysis:

Protein identity was determined, and purity estimated, by SDS-PAGE. Analiquot of 5 μg of each sample was diluted in SDS-PAGE sample bufferwith or without 100 mM DTT as a reductant. The samples were boiled for 5minutes and loaded onto a 10% SDS polyacrylamide gel (LifeGels; French'sForrest, New South Wales, AUS) and electrophoresed. The gel was stainedwith Coomassie R-250 (Bio-Rad; Hercules, Calif.) to visualize proteinbands. For western blot, 0.5 μg/lane total protein was electrophoresedas described above and the gels were then electro-transferred to a PVDFmembrane and blocked with 5% (w/v) dry milk before probing withanti-flagellin antibody (Inotek; Beverly, Mass.) or serum from miceimmunized with a Pam3Cys.H1C1 peptide (SEQ ID NO: 358). After probingwith alkaline phosphatase-conjugated secondary antibodies (Pierce;Rockland, Ill.), protein bands were visualized with an alkalinephosphatase chromogenic substrate (Promega; Madison, Wis.).

Results and Discussion

The purified and refolded STF2.1×H1C1 protein (SEQ ID NO: 342) was foundto be monomeric as judged by the elution profile on a Superdex 200 gelfiltration column. The majority of the protein was found in the includedvolume with the major peak eluting at approximately 14 mls. Thiscorresponds very closely with the known elution profile of purified,monomeric flagellin on this column. Almost no protein was seen elutingin the void volume, known to be approximately 7 mls for this column,demonstrating that virtually no aggregates are present.

Immunogenicity and Efficacy of Pam3Cys.H1C1, a Fusion Peptide ComprisingPam3Cys (TLR2Agonist) and the Maturational Cleavage Site Peptide ofInfluenza A Hemagglutinin Materials and Methods

Peptide Design and Synthesis:

Pam3 (tri-palmoytyl) is the natural ligand for Toll-like Receptor 2(TLR2), and it is natively expressed as the lipidation motif ofbacterial lipoprotein (BLP, SEQ ID NO: 357). Pam3 can be made bychemical synthesis and conjugated to macromolecules such as proteins orcoupled to the N-terminus of synthetic peptides using standard peptidesynthesis chemistry. This strategy usually involves the synthesis of apeptide of interest concluded by the coupling of Pam3-modified cysteine(Pam3Cys) to the amino terminus to yield the lipopeptide of interest.This approach was used to synthesize a lipidated HA cleavage fragmentpeptide, Pam3Cys.H1C1 (Pam3Cys-SLWSEENIPSIQSRGLFGAIAGFIEE, SEQ ID NO:358).

Mice and Immunization:

Female BALB/c mice (Jackson Laboratory, Bar Harbor, Me.) were used atthe age of 6-8 weeks. Mice were divided into groups of 10 and receivedinguinal subcutaneous (s.c) immunizations on days 0 and 14 as follows:

-   8) PBS (phosphate buffered saline).-   9) 20 μg of H1C1 native peptide (SEQ ID NO: 358) in saline buffer    (10 mM Histidine, 10 mM Tris, 75 mM NaCl, 5% (vol/vol) sucrose,    0.02% (w/v) Polysorbate-80, 0.1 mM EDTA, 0.5% (v/v) ethanol, pH 7.2)-   10) 20 μg of Pam3Cys.H1C1 peptide (SEQ ID NO: 358) in saline buffer

An additional group of five mice received an experimentally determinedsublethal challenge with 8×10¹ egg infectious dosages (EID) PR/8/34 andwere allowed to convalesce for >21 days. These animals were then used asimmune convalescent positive controls during the challenge studies. Micewere bled on days 10 (primary) and 21 (boost), and sera were clarifiedby clotting and centrifugation and stored at −20° C.

Serum Antibody Determination:

H1C1-specific IgG levels were determined by ELISA. 96-well ELISA plates(Costar (Cat #9018) Corning, N.Y.) were coated overnight at 4° C. with100 μl/well H1C1 peptide (SEQ ID NO: 358) in PBS (5 μg/ml). Plates wereblocked with 200 μl/well of Assay Diluent Buffer (ADB; BD Pharmingen,(Cat#: 555213) (San Diego, Calif.) for one hour at room temperature. Theplates were washed three times in PBS+0.05% (v/v) Tween 20 (PBS-T).Dilutions of the sera in ADB were added (100 μl/well) and the plateswere incubated overnight at 4° C. The plates were washed three timeswith PBS-T. HRP-labeled goat anti-mouse IgG antibodies (JacksonImmunochemical, West Grove, Pa. (Cat#: 115-035-146)) diluted in ADB wereadded (100 μl/well) and the plates were incubated at room temperaturefor 1 hour. The plates were washed three times with PBS-T. After addingTMB Ultra substrate (Pierce (Cat 34028), Rockford, Ill.)) and monitoringcolor development, A₄₅₀ was measured on a Tecan Farcyte (Durham, N.C.)microplate spectrophotometer.

Influenza Virus Challenge of Mice.

To assess efficacy, mice immunized as described above were challenged onday 28 by intranasal administration of an LD₉₀ (dose lethal to 90% ofmice) (8×10³ EID) of influenza A isolate PR/8/34. Animals were monitoreddaily for 21 days following the challenge for survival, weight loss andclinical presentation. The % weight loss was calculated based on themean of ((Daily weight (g)/Initial weight (g) day 28)×100) of eachindividual animal per group. Clinical scores were assigned as follows: 4pts=healthy, 3 pts=reduced grooming, 2 pts=reduced physical activity and1 pt=moribund. (Experimental results for clinical scores and weight lossreflect the results based on surviving animals on the day evaluated).

Results and Discussion

Induction of H1C1-Specific Antibody Responses Following Immunizationwith Pam3Cys.H1C1:

Mice were immunized with native H1C1 peptide (SEQ ID NO: 358) and thesame peptide modified by the addition of an amino terminal Pam3Cysresidue, to test the hypothesis that linkage of the peptide to a TLR2ligand would increase its immunogenicity. Immunogenicity was determinedby measuring levels of antibodies to native H1C1 (SEQ ID NO: 358) in thesera of the immunized mice. The results show clearly that mice immunizedwith the Pam3Cys-modified peptide generated higher antibody titers tothe immunizing peptide backbone sequence than did mice immunized withthe native peptide (SEQ ID NO: 358) (FIG. 27).

Protection from Lethal Influenza Virus Challenge Following Immunizationwith Pam3Cys.H1C1

The results in FIG. 27 demonstrated that immunization of mice withPam3Cys.H1C1 generated an antibody response that recognized native H1C1peptide. In order to evaluate efficacy, the same mice were challenged onday 28 with an LD₉₀ (8×10³EID) of PR/8/34 virus administeredintra-nasally. Mice were monitored daily for 21 days following thechallenge for survival, weight loss and clinical presentation. As shownin FIG. 28, PBS-immunized mice died between 6 and 10 dayspost-challenge, while convalescent mice survived beyond the 21-dayobservation period. Mice immunized with native H1C1 (SEQ ID NO: 358)began to die by day 6 post-infection at a rate similar to the PBScontrol mice, although 2 of the 10 mice survived beyond the 21-dayobservation period. In contrast, and similar to the convalescent mice,all mice immunized with Pam3Cys.H1C1 survived beyond the 21-dayobservation period. Thus, while H1C1 alone is not potently immunogenic(FIG. 27) nor protective (FIG. 28), the same sequence coupled to a TLR2ligand is both immunogenic and protective.

STF2.blp SEQ ID NO: 340ATGGCACAAGTAATCAACACTAACAGTCTGTCGCTGCTGACCCAGAATAACCTGAACAAATCCCAGTCCGCACTGGGCACCGCTATCGAGCGTCTGTCTTCTGGTCTGCGTATCAACAGCGCGAAAGACGATGCGGCAGGTCAGGCGATTGCTAACCGTTTCACCGCGAACATCAAAGGTCTGACTCAGGCTTCCCGTAACGCTAACGACGGTATCTCCATTGCGCAGACCACTGAAGGCGCGCTGAACGAAATCAACAACAACCTGCAGCGTGTGCGTGAACTGGCGGTTCAGTCTGCTAACAGCACCAACTCCCAGTCTGACCTCGACTCCATCCAGGCTGAAATCACCCAGCGCCTGAACGAAATCGACCGTGTATCCGGCCAGACTCAGTTCAACGGCGTGAAAGTCCTGGCGCAGGACAACACCCTGACCATCCAGGTTGGCGCCAACGACGGTGAAACTATCGATATCGATCTGAAGCAGATCAACTCTCAGACCCTGGGTCTGGACTCACTGAACGTGCAGAAAGCGTATGATGTGAAAGATACAGCAGTAACAACGAAAGCTTATGCCAATAATGGTACTACACTGGATGTATCGGGTCTTGATGATGCAGCTATTAAAGCGGCTACGGGTGGTACGAATGGTACGGCTTCTGTAACCGGTGGTGCGGTTAAATTTGACGCAGATAATAACAAGTACTTTGTTACTATTGGTGGCTTTACTGGTGCTGATGCCGCCAAAAATGGCGATTATGAAGTTAACGTTGCTACTGACGGTACAGTAACCCTTGCGGCTGGCGCAACTAAAACCACAATGCCTGCTGGTGCGACAACTAAAACAGAAGTACAGGAGTTAAAAGATACACCGGCAGTTGTTTCAGCAGATGCTAAAAATGCCTTAATTGCTGGCGGCGTTGACGCTACCGATGCTAATGGCGCTGAGTTGGTCAAAATGTCTTATACCGATAAAAATGGTAAGACAATTGAAGGCGGTTATGCGCTTAAAGCTGGCGATAAGTATTACGCCGCAGATTACGATGAAGCGACAGGAGCAATTAAAGCTAAAACTACAAGTTATACTGCTGCTGACGGCACTACCAAAACAGCGGCTAACCAACTGGGTGGCGTAGACGGTAAAACCGAAGTCGTTACTATCGACGGTAAAACCTACAATGCCAGCAAAGCCGCTGGTCATGATTTCAAAGCACAACCAGAGCTGGCGGAAGCAGCCGCTAAAACCACCGAAAACCCGCTGCAGAAAATTGATGCCGCGCTGGCGCAGGTGGATGCGCTGCGCTCTGATCTGGGTGCGGTACAAAACCGTTTCAACTCTGCTATCACCAACCTGGGCAATACCGTAAACAATCTGTCTGAAGCGCGTAGCCGTATCGAAGATTCCGACTACGCGACCGAAGTTTCCAACATGTCTCGCGCGCAGATTCTGCAGCAGGCCGGTACTTCCGTTCTGGCGCAGGCTAACCAGGTCCCGCAGAACGTGCTGAGCCTGTTACGT HA maturation cleavage fragment (H1N1) SEQ ID NO: 341NIPSIQSRGLFGAIAGFIE STF2.1xH1C1 SEQ ID NO: 342MAQVINTNSLSLLTQNNLNKSQSALGTAIERLSSGLRINSAKDDAAGQAIANRFTANIKGLTQASRNANDGISIAQTTEGALNEINNNLQRVRELAVQSANSTNSQSDLDSIQAEITQRLNEIDRVSGQTQFNGVKVLAQDNTLTIQVGANDGETIDIDLKQINSQTLGLDSLNVQKAYDVKDTAVTTKAYANNGTTLDVSGLDDAAIKAATGGTNGTASVTGGAVKFDADNNKYFVTIGGFTGADAAKNGDYEVNVATDGTVTLAAGATKTTMPAGATTKTEVQELKDTPAVVSADAKNALIAGGVDATDANGAELVKMSYTDKNGKTIEGGYALKAGDKYYAADYDEATGAIKAKTTSYTAADGTTKTAANQLGGVDGKTEVVTIDGKTYNASKAAGHDFKAQPELAEAAAKTTENPLQKIDAALAQVDALRSDLGAVQNRFNSAITNLGNTVNNLSEARSRIEDSDYATEVSNMSRAQILQQAGTSVLAQANQVPQNVLSLLAMEWENIPSIQSRGLFGAIAGFIE STF2.2xH1C1 SEQ ID NO:343 MAQVINTNSLSLLTQNNLNKSQSALGTAIERLSSGLRINSAKDDAAGQAIANRFTANIKGLTQASRNANDGISIAQTTEGALNEINNNLQRVRELAVQSANSTNSQSDLDSIQAEITQRLNEIDRVSGQTQFNGVKVLAQDNTLTIQVGANDGETIDIDLKQINSQTLGLDSLNVQKAYDVKDTAVTTKAYANNGTTLDVSGLDDAAIKAATGGTNGTASVTGGAVKFDADNNKYFVTIGGFTGADAAKNGDYEVNVATDGTVTLAAGATKTTMPAGATTKTEVQELKDTPAVVSADAKNALIAGGVDATDANGAELVKMSYTDKNGKTIEGGYALKAGDKYYAADYDEATGAIKAKTTSYTAADGTTKTAANQLGGVDGKTEVVTIDGKTYNASKAAGHDFKAQPELAEAAAKTTENPLQKIDAALAQVDALRSDLGAVQNRFNSAITNLGNTVNNLSEARSRIEDSDYATEVSNMSRAQILQQAGTSVLAQANQVPQNVLSLLAMEWENIPSIQSRGLFGAIAGFIEEWENIPSIQSR GLFGAIAGFIESTF2.3xH1C1 SEQ ID NO: 344MAQVINTNSLSLLTQNNLNKSQSALGTAIERLSSGLRINSAKDDAAGQAIANRFTANIKGLTQASRNANDGISIAQTTEGALNEINNNLQRVRELAVQSANSTNSQSDLDSIQAEITQRLNEIDRVSGQTQFNGVKVLAQDNTLTIQVGANDGETIDIDLKQINSQTLGLDSLNVQKAYDVKDTAVTTKAYANNGTTLDVSGLDDAAIKAATGGTNGTASVTGGAVKFDADNNKYFVTIGGFTGADAAKNGDYEVNVATDGTVTLAAGATKTTMPAGATTKTEVQELKDTPAVVSADAKNALIAGGVDATDANGAELVKMSYTDKNGKTIEGGYALKAGDKYYAADYDEATGAIKAKTTSYTAADGTTKTAANQLGGVDGKTEVVTIDGKTYNASKAAGHDFKAQPELAEAAAKTTENPLQKIDAALAQVDALRSDLGAVQNRFNSAITNLGNTVNNLSEARSRIEDSDYATEVSNMSRAQILQQAGTSVLAQANQVPQNVLSLLAMEWENIPSIQSRGLFGAIAGFIEEWENIPSIQSRGLFGAIAGFIEEWENIPSIQSRGLFGAIAGFIE STF2.4xH1C1 SEQ ID NO: 345MAQVINTNSLSLLTQNNLNKSQSALGTAIERLSSGLRINSAKDDAAGQAIANRFTANIKGLTQASRNANDGISIAQTTEGALNEINNNLQRVRELAVQSANSTNSQSDLDSIQAEITQRLNEIDRVSGQTQFNGVKVLAQDNTLTIQVGANDGETIDIDLKQINSQTLGLDSLNVQKAYDVKDTAVTTKAYANNGTTLDVSGLDDAAIKAATGGTNGTASVTGGAVKFDADNNKYFVTIGGFTGADAAKNGDYEVNVATDGTVTLAAGATKTTMPAGATTKTEVQELKDTPAVVSADAKNALIAGGVDATDANGAELVKMSYTDKNGKTIEGGYALKAGDKYYAADYDEATGAIKAKTTSYTAADGTTKTAANQLGGVDGKTEVVTIDGKTYNASKAAGHDFKAQPELAEAAAKTTENPLQKIDAALAQVDALRSDLGAVQNRFNSAITNLGNTVNNLSEARSRIEDSDYATEVSNMSRAQILQQAGTSVLAQANQVPQNVLSLLAMEWENIPSIQSRGLFGAIAGFIEEWENIPSIQSRGLFGAIAGFIEEWENIPSIQSRGLFGAIAGFIEEWENIPSIQSRGLFGAIAGFIE STF2.4xH1C2 SEQID NO: 346 MAQVINTNSLSLLTQNNLNKSQSALGTAIERLSSGLRINSAKDDAAGQATANRFTANIKGLTQASRNANDGISIAQTTEGALNEINNNLQRVRELAVQSANSTNSQSDLDSIQAEITQRLNEIDRVSGQTQFNGVKVLAQDNTLTIQVGANDGETIDIDLKQINSQTLGLDSLNVQKAYDVKDTAVTTKAYANNGTTLDVSGLDDAAIKAATGGTNGTASVTGGAVKFDADNNKYFVTIGGFTGADAAKNGDYEVNVATDGTVTLAAGATKTTMPAGATTKTEVQELKDTPAVVSADAKNALIAGGVDATDANGAELVKMSYTDKNGKTIEGGYALKAGDKYYAADYDEATGAIKAKTTSYTAADGTTKTAANQLGGVDGKTEVVTIDGKTYNASKAAGHDFKAQPELAEAAAKTTENPLQKIDAALAQVDALRSDLGAVQNRFNSAITNLGNTVNNLSEARSRIEDSDYATEVSNMSRAQILQQAGTSVLAQANQVPQNVLSLLAGCGSEWENIPSIQSRGLFGAIAGFIEEWENIPSIQSRGLFGAIAGFIEEWENIPSIQSRGLFGAIAGFIEEWENIPSIQSRGLFGAIAGFIESG C HAmaturation cleavage fragment (H5N1) SEQ ID NO: 347 RERRRKKRGLFGAIAGFIESTF2.4xH5C1 SEQ ID NO: 348MAQVINTNSLSLLTQNNLNKSQSALGTAIERLSSGLRINSAKDDAAGQAIANRFTANIKGLTQASRNANDGISIAQTTEGALNEINNNLQRVRELAVQSANSTNSQSDLDSIQAEITQRLNEIDRVSGQTQFNGVKVLAQDNTLTIQVGANDGETIDIDLKQINSQTLGLDSLNVQKAYDVKDTAVTTKAYANNGTTLDVSGLDDAAIKAATGGTNGTASVTGGAVKFDADNNKYFVTIGGFTGADAAKNGDYEVNVATDGTVTLAAGATKTTMPAGATTKTEVQELKDTPAVVSADAKNALIAGGVDATDANGAELVKMSYTDKNGKTIEGGYALKAGDKYYAADYDEATGAIKAKTTSYTAADGTTKTAANQLGGVDGKTEVVTIDGKTYNASKAAGHDFKAQPELAEAAAKTTENPLQKIDAALAQVDALRSDLGAVQNRFNSAITNLGNTVNNLSEARSRIEDSDYATEVSNMSRAQILQQAGTSVLAQANQVPQNVLSLLAEWERERRRKKRGLFGAIAGFIEEWERERRRKKRGLFGAIAGFIEEWERERRRKKRGLFGAIAGFIEEWERERRRKKRGLFGAIAGFIE HA maturationcleavage fragment (H3N2) SEQ ID NO: 349 NVPEKQTRGIFGAIAGFIE HAmaturation cleavage fragment (H2N1/H2N2/H2N3/H2N5/H2N8/H2N9) SEQ ID NO:350 NVPQIESRGLFGAIAGFIE HA maturation cleavage fragment (B strain) SEQID NO: 351 PAKLLKERGFFGAIAGFLE STF2.4xH1C1 SEQ ID NO: 352ATGGCACAAGTAATCAACACTAACAGTCTGTCGCTGCTGACCCAGAATAACCTGAACAAATCCCAGTCCGCACTGGGCACCGCTATCGAGCGTCTGTCTTCTGGTCTGCGTATCAACAGCGCGAAAGACGATGCGGCAGGTCAGGCGATTGCTAACCGTTTCACCGCGAACATCAAAGGTCTGACTCAGGCTTCCCGTAACGCTAACGACGGTATCTCCATTGCGCAGACCACTGAAGGCGCGCTGAACGAAATCAACAACAACCTGCAGCGTGTGCGTGAACTGGCGGTTCAGTCTGCTAACAGCACCAACTCCCAGTCTGACCTCGACTCCATCCAGGCTGAAATCACCCAGCGCCTGAACGAAATCGACCGTGTATCCGGCCAGACTCAGTTCAACGGCGTGAAAGTCCTGGCGCAGGACAACACCCTGACCATCCAGGTTGGCGCCAACGACGGTGAAACTATCGATATCGATCTGAAGCAGATCAACTCTCAGACCCTGGGTCTGGACTCACTGAACGTGCAGAAAGCGTATGATGTGAAAGATACAGCAGTAACAACGAAAGCTTATGCCAATAATGGTACTACACTGGATGTATCGGGTCTTGATGATGCAGCTATTAAAGCGGCTACGGGTGGTACGAATGGTACGGCTTCTGTAACCGGTGGTGCGGTTAAATTTGACGCAGATAATAACAAGTACTTTGTTACTATTGGTGGCTTTACTGGTGCTGATGCCGCCAAAAATGGCGATTATGAAGTTAACGTTGCTACTGACGGTACAGTAACCCTTGCGGCTGGCGCAACTAAAACCACAATGCCTGCTGGTGCGACAACTAAAACAGAAGTACAGGAGTTAAAAGATACACCGGCAGTTGTTTCAGCAGATGCTAAAAATGCCTTAATTGCTGGCGGCGTTGACGCTACCGATGCTAATGGCGCTGAGTTGGTCAAAATGTCTTATACCGATAAAAATGGTAAGACAATTGAAGGCGGTTATGCGCTTAAAGCTGGCGATAAGTATTACGCCGCAGATTACGATGAAGCGACAGGAGCAATTAAAGCTAAAACTACAAGTTATACTGCTGCTGACGGCACTACCAAAACAGCGGCTAACCAACTGGGTGGCGTAGACGGTAAAACCGAAGTCGTTACTATCGACGGTAAAACCTACAATGCCAGCAAAGCCGCTGGTCATGATTTCAAAGCACAACCAGAGCTGGCGGAAGCAGCCGCTAAAACCACCGAAAACCCGCTGCAGAAAATTGATGCCGCGCTGGCGCAGGTGGATGCGCTGCGCTCTGATCTGGGTGCGGTACAAAACCGTTTCAACTCTGCTATCACCAACCTGGGCAATACCGTAAACAATCTGTCTGAAGCGCGTAGCCGTATCGAAGATTCCGACTACGCGACCGAAGTTTCCAACATGTCTCGCGCGCAGATTCTGCAGCAGGCCGGTACTTCCGTTCTGGCGCAGGCTAACCAGGTCCCGCAGAACGTGCTGTCTCTGTTAGCGATGGAATGGGAGAACATCCCTAGCATCCAATCTCGCGGCCTGTTTGGCGCTATCGCGGGCTTTATCGAAGAATGGGAGAACATCCCGAGCATCCAATCTCGCGGTCTGTTTGGTGCGATCGCTGGTTTCATCGAGGAGTGGGAGAACATTCCTAGCATTCAAAGCCGTGGCCTGTTCGGCGCTATTGCAGGTTTTATTGAAGAATGGGAAAATATCCCGTCTATCCAATCCCGCGGTCTGTTCGGCGCGATCGCAGGTTTCATTGAATAATAAGCTAAGC STF2.3xH1C1SEQ ID NO: 353ATGGCACAAGTAATCAACACTAACAGTCTGTCGCTGCTGACCCAGAATAACCTGAACAAATCCCAGTCCGCACTGGGCACCGCTATCGAGCGTCTGTCTTCTGGTCTGCGTATCAACAGCGCGAAAGACGATGCGGCAGGTCAGGCGATTGCTAACCGTTTCACCGCGAACATCAAAGGTCTGACTCAGGCTTCCCGTAACGCTAACGACGGTATCTCCATTGCGCAGACCACTGAAGGCGCGCTGAACGAAATCAACAACAACCTGCAGCGTGTGCGTGAACTGGCGGTTCAGTCTGCTAACAGCACCAACTCCCAGTCTGACCTCGACTCCATCCAGGCTGAAATCACCCAGCGCCTGAACGAAATCGACCGTGTATCCGGCCAGACTCAGTTCAACGGCGTGAAAGTCCTGGCGCAGGACAACACCCTGACCATCCAGGTTGGCGCCAACGACGGTGAAACTATCGATATCGATCTGAAGCAGATCAACTCTCAGACCCTGGGTCTGGACTCACTGAACGTGCAGAAAGCGTATGATGTGAAAGATACAGCAGTAACAACGAAAGCTTATGCCAATAATGGTACTACACTGGATGTATCGGGTCTTGATGATGCAGCTATTAAAGCGGCTACGGGTGGTACGAATGGTACGGCTTCTGTAACCGGTGGTGCGGTTAAATTTGACGCAGATAATAACAAGTACTTTGTTACTATTGGTGGCTTTACTGGTGCTGATGCCGCCAAAAATGGCGATTATGAAGTTAACGTTGCTACTGACGGTACAGTAACCCTTGCGGCTGGCGCAACTAAAACCACAATGCCTGCTGGTGCGACAACTAAAACAGAAGTACAGGAGTTAAAAGATACACCGGCAGTTGTTTCAGCAGATGCTAAAAATGCCTTAATTGCTGGCGGCGTTGACGCTACCGATGCTAATGGCGCTGAGTTGGTCAAAATGTCTTATACCGATAAAAATGGTAAGACAATTGAAGGCGGTTATGCGCTTAAAGCTGGCGATAAGTATTACGCCGCAGATTACGATGAAGCGACAGGAGCAATTAAAGCTAAAACTACAAGTTATACTGCTGCTGACGGCACTACCAAAACAGCGGCTAACCAACTGGGTGGCGTAGACGGTAAAACCGAAGTCGTTACTATCGACGGTAAAACCTACAATGCCAGCAAAGCCGCTGGTCATGATTTCAAAGCACAACCAGAGCTGGCGGAAGCAGCCGCTAAAACCACCGAAAACCCGCTGCAGAAAATTGATGCCGCGCTGGCGCAGGTGGATGCGCTGCGCTCTGATCTGGGTGCGGTACAAAACCGTTTCAACTCTGCTATCACCAACCTGGGCAATACCGTAAACAATCTGTCTGAAGCGCGTAGCCGTATCGAAGATTCCGACTACGCGACCGAAGTTTCCAACATGTCTCGCGCGCAGATTCTGCAGCAGGCCGGTACTTCCGTTCTGGCGCAGGCTAACCAGGTCCCGCAGAACGTGCTGAGCCTGTTAGCGATGGAATGGGAAAATATCCCTAGCATCCAATCTCGCGGTCTGTTCGGTGCTATTGCTGGCTTCATCGAGGAATGGGAGAACATCCCATCTATTCAGTCTCGCGGCCTGTTTGGTGCGATCGCGGGTTTTATTGAGGAATGGGAAAACATTCCAAGCATTCAGTCACGTGGTCTTTTCGGCGCCATCGCTGGTTTTATCGAATGATAAGCTTAGCCCAAGG STF2.2xH1C1SEQ ID NO: 354ATGGCACAAGTAATCAACACTAACAGTCTGTCGCTGCTGACCCAGAATAACCTGAACAAATCCCAGTCCGCACTGGGCACCGCTATCGAGCGTCTGTCTTCTGGTCTGCGTATCAACAGCGCGAAAGACGATGCGGCAGGTCAGGCGATTGCTAACCGTTTCACCGCGAACATCAAAGGTCTGACTCAGGCTTCCCGTAACGCTAACGACGGTATCTCCATTGCGCAGACCACTGAAGGCGCGCTGAACGAAATCAACAACAACCTGCAGCGTGTGCGTGAACTGGCGGTTCAGTCTGCTAACAGCACCAACTCCCAGTCTGACCTCGACTCCATCCAGGCTGAAATCACCCAGCGCCTGAACGAAATCGACCGTGTATCCGGCCAGACTCAGTTCAACGGCGTGAAAGTCCTGGCGCAGGACAACACCCTGACCATCCAGGTTGGCGCCAACGACGGTGAAACTATCGATATCGATCTGAAGCAGATCAACTCTCAGACCCTGGGTCTGGACTCACTGAACGTGCAGAAAGCGTATGATGTGAAAGATACAGCAGTAACAACGAAAGCTTATGCCAATAATGGTACTACACTGGATGTATCGGGTCTTGATGATGCAGCTATTAAAGCGGCTACGGGTGGTACGAATGGTACGGCTTCTGTAACCGGTGGTGCGGTTAAATTTGACGCAGATAATAACAAGTACTTTGTTACTATTGGTGGCTTTACTGGTGCTGATGCCGCCAAAAATGGCGATTATGAAGTTAACGTTGCTACTGACGGTACAGTAACCCTTGCGGCTGGCGCAACTAAAACCACAATGCCTGCTGGTGCGACAACTAAAACAGAAGTACAGGAGTTAAAAGATACACCGGCAGTTGTTTCAGCAGATGCTAAAAATGCCTTAATTGCTGGCGGCGTTGACGCTACCGATGCTAATGGCGCTGAGTTGGTCAAAATGTCTTATACCGATAAAAATGGTAAGACAATTGAAGGCGGTTATGCGCTTAAAGCTGGCGATAAGTATTACGCCGCAGATTACGATGAAGCGACAGGAGCAATTAAAGCTAAAACTACAAGTTATACTGCTGCTGACGGCACTACCAAAACAGCGGCTAACCAACTGGGTGGCGTAGACGGTAAAACCGAAGTCGTTACTATCGACGGTAAAACCTACAATGCCAGCAAAGCCGCTGGTCATGATTTCAAAGCACAACCAGAGCTGGCGGAAGCAGCCGCTAAAACCACCGAAAACCCGCTGCAGAAAATTGATGCCGCGCTGGCGCAGGTGGATGCGCTGCGCTCTGATCTGGGTGCGGTACAAAACCGTTTCAACTCTGCTATCACCAACCTGGGCAATACCGTAAACAATCTGTCTGAAGCGCGTAGCCGTATCGAAGATTCCGACTACGCGACCGAAGTTTCCAACATGTCTCGCGCGCAGATTCTGCAGCAGGCCGGTACTTCCGTTCTGGCGCAGGCTAACCAGGTCCCGCAGAACGTGCTGAGCCTGTTAGCGATGGAATGGGAAAATATCCCTAGCATCCAATCTCGCGGTCTGTTCGGTGCTATTGCTGGCTTCATCGAGGAATGGGAGAACATCCCATCTATTCAGTCTCGCGGCCTGTTTGGTGCGATCGCGGGTTTTATTGAGTGATAAGCTTAGCCCAAGG STF2.1xH1C1 SEQ IDNO: 355 ATGGCACAAGTAATCAACACTAACAGTCTGTCGCTGCTGACCCAGAATAACCTGAACAAATCCCAGTCCGCACTGGGCACCGCTATCGAGCGTCTGTCTTCTGGTCTGCGTATCAACAGCGCGAAAGACGATGCGGCAGGTCAGGCGATTGCTAACCGTTTCACCGCGAACATCAAAGGTCTGACTCAGGCTTCCCGTAACGCTAACGACGGTATCTCCATTGCGCAGACCACTGAAGGCGCGCTGAACGAAATCAACAACAACCTGCAGCGTGTGCGTGAACTGGCGGTTCAGTCTGCTAACAGCACCAACTCCCAGTCTGACCTCGACTCCATCCAGGCTGAAATCACCCAGCGCCTGAACGAAATCGACCGTGTATCCGGCCAGACTCAGTTCAACGGCGTGAAAGTCCTGGCGCAGGACAACACCCTGACCATCCAGGTTGGCGCCAACGACGGTGAAACTATCGATATCGATCTGAAGCAGATCAACTCTCAGACCCTGGGTCTGGACTCACTGAACGTGCAGAAAGCGTATGATGTGAAAGATACAGCAGTAACAACGAAAGCTTATGCCAATAATGGTACTACACTGGATGTATCGGGTCTTGATGATGCAGCTATTAAAGCGGCTACGGGTGGTACGAATGGTACGGCTTCTGTAACCGGTGGTGCGGTTAAATTTGACGCAGATAATAACAAGTACTTTGTTACTATTGGTGGCTTTACTGGTGCTGATGCCGCCAAAAATGGCGATTATGAAGTTAACGTTGCTACTGACGGTACAGTAACCCTTGCGGCTGGCGCAACTAAAACCACAATGCCTGCTGGTGCGACAACTAAAACAGAAGTACAGGAGTTAAAAGATACACCGGCAGTTGTTTCAGCAGATGCTAAAAATGCCTTAATTGCTGGCGGCGTTGACGCTACCGATGCTAATGGCGCTGAGTTGGTCAAAATGTCTTATACCGATAAAAATGGTAAGACAATTGAAGGCGGTTATGCGCTTAAAGCTGGCGATAAGTATTACGCCGCAGATTACGATGAAGCGACAGGAGCAATTAAAGCTAAAACTACAAGTTATACTGCTGCTGACGGCACTACCAAAACAGCGGCTAACCAACTGGGTGGCGTAGACGGTAAAACCGAAGTCGTTACTATCGACGGTAAAACCTACAATGCCAGCAAAGCCGCTGGTCATGATTTCAAAGCACAACCAGAGCTGGCGGAAGCAGCCGCTAAAACCACCGAAAACCCGCTGCAGAAAATTGATGCCGCGCTGGCGCAGGTGGATGCGCTGCGCTCTGATCTGGGTGCGGTACAAAACCGTTTCAACTCTGCTATCACCAACCTGGGCAATACCGTAAACAATCTGTCTGAAGCGCGTAGCCGTATCGAAGATTCCGACTACGCGACCGAAGTTTCCAACATGTCTCGCGCGCAGATTCTGCAGCAGGCCGGTACTTCCGTTCTGGCGCAGGCTAACCAGGTCCCGCAGAACGTGCTGAGCCTGTTAGCGATGGAATGGGAAAATATCCCTAGCATCCAATCTCGCGGTCTGTTCGGTGCTATTGCTGGCTTCATCGAGTGATAAGCTTAGCCCAAGG STF2.4xH1C1 SEQ ID NO:356 ATGGCACAAGTAATCAACACTAACAGTCTGTCGCTGCTGACCCAGAATAACCTGAACAAATCCCAGTCCGCACTGGGCACCGCTATCGAGCGTCTGTCTTCTGGTCTGCGTATCAACAGCGCGAAAGACGATGCGGCAGGTCAGGCGATTGCTAACCGTTTCACCGCGAACATCAAAGGTCTGACTCAGGCTTCCCGTAACGCTAACGACGGTATCTCCATTGCGCAGACCACTGAAGGCGCGCTGAACGAAATCAACAACAACCTGCAGCGTGTGCGTGAACTGGCGGTTCAGTCTGCTAACAGCACCAACTCCCAGTCTGACCTCGACTCCATCCAGGCTGAAATCACCCAGCGCCTGAACGAAATCGACCGTGTATCCGGCCAGACTCAGTTCAACGGCGTGAAAGTCCTGGCGCAGGACAACACCCTGACCATCCAGGTTGGCGCCAACGACGGTGAAACTATCGATATCGATCTGAAGCAGATCAACTCTCAGACCCTGGGTCTGGACTCACTGAACGTGCAGAAAGCGTATGATGTGAAAGATACAGCAGTAACAACGAAAGCTTATGCCAATAATGGTACTACACTGGATGTATCGGGTCTTGATGATGCAGCTATTAAAGCGGCTACGGGTGGTACGAATGGTACGGCTTCTGTAACCGGTGGTGCGGTTAAATTTGACGCAGATAATAACAAGTACTTTGTTACTATTGGTGGCTTTACTGGTGCTGATGCCGCCAAAAATGGCGATTATGAAGTTAACGTTGCTACTGACGGTACAGTAACCCTTGCGGCTGGCGCAACTAAAACCACAATGCCTGCTGGTGCGACAACTAAAACAGAAGTACAGGAGTTAAAAGATACACCGGCAGTTGTTTCAGCAGATGCTAAAAATGCCTTAATTGCTGGCGGCGTTGACGCTACCGATGCTAATGGCGCTGAGTTGGTCAAAATGTCTTATACCGATAAAAATGGTAAGACAATTGAAGGCGGTTATGCGCTTAAAGCTGGCGATAAGTATTACGCCGCAGATTACGATGAAGCGACAGGAGCAATTAAAGCTAAAACTACAAGTTATACTGCTGCTGACGGCACTACCAAAACAGCGGCTAACCAACTGGGTGGCGTAGACGGTAAAACCGAAGTCGTTACTATCGACGGTAAAACCTACAATGCCAGCAAAGCCGCTGGTCATGATTTCAAAGCACAACCAGAGCTGGCGGAAGCAGCCGCTAAAACCACCGAAAACCCGCTGCAGAAAATTGATGCCGCGCTGGCGCAGGTGGATGCGCTGCGCTCTGATCTGGGTGCGGTACAAAACCGTTTCAACTCTGCTATCACCAACCTGGGCAATACCGTAAACAATCTGTCTGAAGCGCGTAGCCGTATCGAAGATTCCGACTACGCGACCGAAGTTTCCAACATGTCTCGCGCGCAGATTCTGCAGCAGGCCGGTACTTCCGTTCTGGCGCAGGCTAACCAGGTCCCGCAGAACGTGCTGTCTCTGTTAGCGGGTTGTGGTTCCGAGTGGGAAAATATTCCGTCTATCCAGAGCCGTGGTCTGTTCGGCGCAATTGCTGGCTTCATTGAAGAATGGGAAAACATCCCGTCCATCCAGAGCCGTGGCCTGTTCGGCGCCATTGCTGGTTTCATCGAGGAATGGGAAAACATTCCGTCCATCCAGTCCCGCGGTCTGTTTGGCGCTATCGCCGGTTTCATTGAGGAATGGGAAAATATCCCTTCCATCCAGTCTCGTGGTCTGTTCGGCGCGATTGCAGGCTTTATCGAATCTGGTTGCTAATAAGCTAAGC Bacterial lipoprotein of E. coli SEQ ID NO: 357MKATKLVLGAVILGSTLLAGCSSNAKIDQLSSDVQTLNAKVDQLSNDVNAMRSDVQAAKDDAARANQRLDNMATKYRK H1C1 native peptide SEQ ID NO: 358 SLWSEENIPSIQSRGLFGAIAGFIEE

Example 15 Flagellin-M2e Fusion Proteins

M2e is conserved across multiple influenza A subtypes (also referred toherein as “strain”). M2e is at least a portion of the M2 protein, inparticular, a 24 amino-terminus (also referred to herein as an“ectodomain”) of the M2 protein. The M2 ectodomain is relatively smallamino acid sequence (24 amino acids) compared to HA (about 566 aminoacids) and NA (about 469 amino acids). The M2e sequence of exemplaryavian influenza A isolates differs from that of human isolates, but ishighly-conserved among the avian isolates (see, for example, SEQ ID NOS:544-556, 570 and 573-578). Four tandem copies of M2e fused to thecarboxy terminus of a flagellin STF2 (full-length or STF2 hingeregion-deleted) were generated. The STF2 without the hinge region isalso referred to herein as “STF2Δ.”

Construction of Fusion Protein

The carboxy-terminal fusion of the synthetic 4×M2e sequence (4consecutive 24 amino acid sequences) with STF2 was constructed asfollows. The pET24A vector was purchased from Novagen, San Diego, Calif.The strategy employed the Seamless Cloning Kit (Catalog number 214400)from Stratagene (La Jolla, Calif. www.stratagene.com) performed by DNA2.0 Inc. (Menlo Park, Calif.). The gene encoding the fusion protein wasin pDrive 4×M2e G00448 and was used as a PCR template for insertpreparation for construction of the C-terminal fusion expressionconstruct with STF2. The synthetic 4×M2e construct pDrive 4×M2e G00448was used as a template for PCR as outlined in the Seamless Cloning Kit(Catalog number 214400) from Stratagene (La Jolla, Calif.). The expectedproduct from this amplification includes the 318 by and the restrictionenzyme sites incorporated into the oligonucleotides used to amplify thisinsert. The procedure was as follows:

PCR Conditions

1 μL-20 ng of pDrive 4×M2e G00448

5 μL of 10× cloned Pfu polymerase buffer

1 μL of 40 mM dNTP mix

10 μL-10 pmol of forward primer 4×M2eforbs1

10 μL-10 pmol of reverse primer 4×M2erevwsto

40 μL ddH₂O

Immediately before starting the thermal cycling 1 μL of PfuTurbo DNA

Polymerase the following were added.

4xM2eforbs1 primer sequence: (SEQ ID NO: 566)5′-CGCTCTTCAMTGAGCTTGCTGACTGAGGTTGAGACCCCGATTC 4xM2erevwsto primersequence: (SEQ ID NO: 567)5′-CGCTCTTCACGCTTATTATCTAGACGGGTCTGAGCTATCGTTAGAG CGAG

This reaction was cycled as follows on a Thermo Hybaid PxE thermalcycler (Waltham, Mass.).

Initial Cycle

Temperature Duration 95°  3 minutes 65° 1 minute 72° 1 minute

Subsequent Nine Cycles

Temperature Duration 95° 45 seconds 65° 35 seconds 72° 1 minute

At this point the following was added to each reaction.

5 μL of 10× cloned Pfu polymerase buffer

1 μL of 5-methyl dNTP mix

44 μL ddH₂O

Subsequently the following thermal cycling was repeated five times.

Temperature Duration 95° 45 seconds 65° 35 seconds 72° 1 minute

The 100 μL product was brought to a volume of 300 μL by the addition ofTE buffer. The resulting product was phenol chloroform (InvitrogenCarlsbad, Calif.—Catalog number 15593-031) extracted once and chloroformextracted once. The amplification product was then ethanol precipitatedby addition of 30 μL of Sodium acetate buffer pH 5.2 and 750 μL of 100%Ethanol. The DNA pellet was washed twice with 300 μL 70% Ethanol allowedto air dry for ten minutes and then resuspended in 50 μL TE buffer.

Amplification of Vector STF2 in pET24.

The previously constructed pET24a/STF2.M2e construct was used as atemplate for PCR as outlined in the Seamless Cloning Kit (Catalog number214400) from Stratagene (La Jolla, Calif.). The expected product fromthis amplification includes the whole of the pET24 plasmid plus the STF2sequences but does not include the single copy of M2E that exists inthis construct. The procedure was as follow:

PCR Conditions

1 μL-40 ng of STF2.M2E pET22-2

5 μL of 10× cloned Pfu polymerase buffer

1 μL of 40 mM dNTP mix

1 μL-10 pmol of primer 4×MECpET24

1 μL-10 pmol of primer 4×M2eC-STF2

40 μL ddH₂O

Immediately before starting the thermal cycling the following wereadded:

1 μL of PfuTurbo DNA Polymerase

4xMECpET24 primer sequence: (SEQ ID NO: 568)5′-GCTCTTCAGCGGCTGAGCAATAACTAGCATAACCCCTTGGG 4xM2eC-STF2 primersequence: (SEQ ID NO: 569) 5′-CGCTCTTCACAGACGTAACAGAGACAGCACGTTCTGCGG

This reaction was cycled as follows on a Thermo Hybaid PxE thermalcycler (Waltham, Mass.).

Initial Cycle

Temperature Duration 95°  3 minutes 65° 1 minute 72° 18 minutes

Subsequent Nine Cycles

Temperature Duration 95° 45 seconds 65° 35 seconds 72° 18 minutes

At this point the following was added to each reaction.

5 μL of 10× cloned Pfu polymerase buffer

1 μL of 5-methyl dNTP mix

44 μL ddH₂O

Subsequently the following thermal cycling was repeated five times.

Temperature Duration 95° 45 seconds 65° 35 seconds 72° 18 minutes

The 100 μL product was brought to a volume of 300 μL by the addition ofTE buffer. The resulting product was phenol chloroform (InvitrogenCarlsbad, Calif.—Catalog number 15593-031) extracted once and chloroformextracted once. The amplification product was then ethanol precipitatedby addition of 30 μL of Sodium acetate buffer pH 5.2 and 750 μL of 100%Ethanol. The DNA pellet was washed twice with 300 μL 70% Ethanol allowedto air dry for ten minutes and then resuspended in 50 μL TE buffer.

Digestion and Ligation of Vector and Insert Amplifications

Eam 1104 I digests were set up separately for vector and insert asfollows:

30 μL of amplified product after ethanol precipitation

5 μL of 10× Universal buffer (Supplied with Seamless Cloning Kit)

4 μL Eam 1104 I restriction enzyme (Supplied with Seamless Cloning Kit)

11 μL ddH₂O

Digests were mixed gently and incubated at 37° C. for one hour andligation reactions of vector and insert products were prepared as aboveperformed as follows (Reagents supplied with Seamless Cloning Kit):

Ingredients added in order listed:

9 μL ddH₂O

5 μL of Eam 1104 I digested 4×M2e amplified insert

5 μL of Eam 1104 I digested STF2.M2E pET22-2 amplified vector

2 μL 10× Ligase buffer

2 μL 10 mM rATP

1 μL T4 DNA Ligase (diluted from stock 1:16)

1 μL Eam 1104 I restriction enzyme

The ligation reactions were mixed gently and incubated for 30 minutes at37° C. The ligations were then stored on ice until transformed intoXL-10 competent cells (Stratagene Catalog number 200314) later than sameday.

Transformation of Ligation into XL-10 Competent Cells

Eppendorf tubes were chilled for ten minutes while the XL-10 (StratageneCatalog number 200314) competent cells thawed on ice.

50 μL of competent cells were aliquoted from the stock tube perligation.

2 μL of β-mercaptoethanol stock which is provided with the XL-10 cells.

This mixture was incubated for ten minutes on ice gently mixing every 2minutes. Seamless cloning ligation reaction (4 μl) was added, swirledgently and then incubated on ice for 30 minutes. The tubes were heatshocked for 35 seconds at 42° C. in a water bath. The tubes wereincubated on ice for at least two minutes. SOC medium (400 μL) wereadded to the cells and incubated for one hour at 37° C. with agitation.

Two LB agar kanamycin (50 m/mL) plates are used to plate 200 μL and 10μL of the transformed cells and allowed to grow overnight.

Screening of Kanamycin Resistant Clones

Recombinant candidates were grown up for minipreps in Luria Brothcontaining Kanamycin (25 ug/mL) and extracted using the QIAprep SpinMiniprep Kit (Qiagen Valencia, Calif. Catalog Number 27106). Candidateclones were screened by restriction enzymes (New England BiolabsBeverly, Mass.) and positive clones were grown up in 100 mL of LuriaBroth containing kanamycin (25 ug/mL) and extracted using the QiagenHiSpeed Plasmid Midi Kit (Catalog number 12643). These clones weresubmitted to GENEWIZ (North Brunswick, N.J.) for sequencing.

Production and Purification of STF2.4×M2e Fusion Protein

STF2.4×M2e in E. coli BLR(DE3)pLysS host (Novagen, San Diego, Calif.,Catalog #69053) was retrieved from glycerol stock and scaled up to 5 L.Cells were grown in LB medium containing 15 μg/ml Kanamycin and 12.5μg/ml Teteracycline to OD₆₀₀=0.4 and induced with 1 mM IPTG for 3 h at37° C. The cells were harvested by centrifugation (7000 rpm×7 minutes ina Sorvall RC5C centrifuge) and resuspended in 2×PBS, 1% glycerol, DNAse,1 mM PMSF, protease inhibitor cocktail and 1 mg/ml lysozyme. Thesuspension was passed through a microfluidizer to lyse the cells. Thelysate was centrifuged (45,000 g for one hour in a Beckman Optima Lultracentrifuge) to separate the soluble fraction from inclusion bodies.Protein was detected by SDS-PAGE in the soluble and insoluble fractions.

The soluble fraction was applied to Sepharose Q resin in the presence ofhigh salt via batch method to reduce DNA, endotoxin, and othercontaminants. The flow through containing the protein of interest wasloaded onto 30 ml Q Sepharose column (Amersham Biosciences). Boundprotein was eluted using a linear gradient from Buffer A to B. (BufferA: 100 mM Tris-Cl, pH 8.0. Buffer B: 100 mM Tris-Cl, 1 M NaCl, pH 8.0).Eluted protein was further purified using a 45 ml Source Q column thatprovided greater resolution needed to resolve contaminating proteins.Bound protein was eluted with a linear gradient from Buffer A to B(Buffer A: 100 mM Tris-C1, pH 8.0 Buffer B: 100 mM Tris-Cl, 1 M NaCl, pH8.0).

Final purification of protein was completed using Superdex-200 gelfiltration chromatography. The column was developed with 100 mM Tris,150 mM NaCl and 1% glycerol plus 1% Na-deoxycholate to remove the LPS.Buffer exchange was carried out using overnight dialysis against buffercontaining 50 mM Tris, 100 mM NaCl and 1% glycerol was done to removeNa-deoxycholate. Protein concentration was determined by the MicroBCAProtein Assay Reagent Kit (Pierce Biotechnology). Purified preparationsof STF2.4×M2e yielded a single band visible with Coomassie stain thatmigrated with an apparent molecular weight of about 64 kDa on 12% SDSpolyacrylamide gels.

Example 16 Expression and Purification of Flagellin (STF2 and STF2Δ)Fusion Protein Constructs Encoding Influenza A M2 Ectodomain Sequences

The consensus M2e sequences from several influenza A strains of humanand avian origin are depicted in SEQ ID NOS: 544-556, 570 and 573-578.To facilitate the cloning of the M2e sequence, two vector cassettes,pMT/STF2 and pMT/STF2Δ, each containing a multiple cloning site (MCS)were generated (See FIGS. 17A and 17B). To generate pMT/STF2, the 1.5 kbgene encoding full length flagellin of Salmonella typhimurium fljb type2 or STF2, was fused to the Ig binding protein (BIP) secretion signal ofpMTBIP/V5-His vector (Invitrogen Corporation, Carlsbad, Calif.) forexpression in Drosophila. The BiP sequence is included at the 5′ end ofthe construct as a secretion signal for expression in Drosophila. Achemically-synthesized 4×M2e gene representing the H1, H2 and H3consensus sequence, SEQ ID NO: 544, was cloned into the MCS of pMT/STF2to create pMT/STF2.4×M2e(H1).

A similar strategy prophetically is employed to clone two H5-associatedM2e sequences, SLLTEVETPTRNEWECRCSDSSDP (SEQ ID NO: 553) (A/VietNam/1203/2004) and SLLTEVETLTRNGWGCRCSDSSDP (SEQ ID NO: 552) (A/HongKong/156/97). Codon-optimized chemically synthesized genes containingfour tandemly repeated copies of the indicated H5-associated M2esequence prophetically are cloned into pMT/STF2 to generateSTF2.4×M2e(H5VN) and STF2.4×M2e(H5HK), respectively. To generate aconstruct that contains multiple M2e forms, the heterologous 4×M2esequence(s) prophetically are inserted into either of the primaryconstructs.

“Heterologous sequences,” as used herein, means sequences from differentspecies. For example, the H1 sequence is a human sequence and the H5sequence is an avian sequence. Thus, the H1 and H5 sequences areheterologous sequences (e.g.,SLLTEVETPTRNEWESRSSDSSDPLESLLTEVETPTRNEWESRSSDSSDPESSLLTEVETPTRNEWESRSSDSSDPGSSLLTEVETPTRNEWESRSSDSSDP (SEQ ID NO: 597), encodedbytctctgctgactgaagtagaaactccaacgcgtaatgaatgggaatcccgttctagcgactcctctgatcctctcgagtccctgctgacggaggttgaaaccccgacccgcaacgagtgggaaagccgttectccgattcctctgatccggagagcagcctgctgaccgaggtagaaaccccgacccgtaatgagtgggaatctcgctcctctgattcttctgacccgggatcctctctgctgaccgaagtggagactccgactcgcaacgaatgggagagccgttcttctgactcctctgacccg (SEQ ID NO:598).

Primary constructs comprise at least one pathogen-associated molecularpattern (e.g., STF2, STF2Δ) and at least a portion of at least oneintegral membrane protein (e.g., M2e, such as SEQ ID NOS: 510 and 544).If there is more than one integral membrane in a primary construct, theintegral membrane proteins are from the same species.

A heterologous construct includes at least two integral membraneproteins such as H1 (human) and H5 (avian), for example, in SEQ ID NOS:583 and 584.

To generate pMT/STF2Δ, the hyper-variable region that spans amino acids170 to 415 of the full-length flagellin gene of SEQ ID NO: 499 wasdeleted and replaced with a short (10 amino acid) flexible linker(GAPVDPASPW, SEQ ID NO: 594) designed to facilitate interactions of theamino and carboxy terminal sequences necessary for TLR5 signaling. Theprotein expressed from this construct retains potent TLR5 activitywhether expressed alone or in fusion with test antigen. Thus, a secondseries of M2e constructs prophetically is generated based on pMT/STF2Δ.Drosophila Dme1-2 cells (Invitrogen Corporation, Carlsbad, Calif.) grownat room temperature in Schneider's medium supplemented with 10% FBS andantibiotics prophetically is transfected with the constructs describedabove using Cellfectin reagent (Invitrogen) according to themanufacturer's instructions. Twenty-four hours post transfection, cellsprophetically is induced with 0.5 mM CuSO₄ in medium lacking FBS andincubated for an additional 48 hours. Conditioned media (CM)prophetically is harvested from induced cultures and screened forprotein expression by SDS-PAGE and Western blot analyses usinganti-flagellin and anti-M2e specific antibodies. The identity, TLRbioactivity of the fusion protein, antigenicity assessed by ELISA and invivo mouse studies for immunogenicity prophetically is performed.

Example 17 Construction and Expression of Flagellin-Hemaglutinin (HA)Constructs

The gene encoding HA from genomic DNA from the in-house laboratorystrain PR8, an attenuated derivative of A/Puerto Rico/8/34 was isolated(SEQ ID NO: 565, encoding SEQ ID NO: 564). The gene was fused to theSTF2Δ cassette that has been previously constructed in pPICZΔ generatingSTF2Δ.HAPR8 (SEQ ID NO: 560, encoding SEQ ID NO: 559) (See FIG. 42).Purified recombinant protein was tested for immunogenicity and efficacyin BALB/c mice. The gene encoding H5N1 of the A/Vietnam/1203/04 strainwas custom synthesized and fused to STF2Δ cassette generating STF2Δ.HAH5(SEQ ID NO: 558, encoding SEQ ID NO: 557). Both human and avian HAconstructs were transformed into Pichia pastoris strains GS 115 and X-33(Invitrogen Corporation, Carlsbad, Calif.). Selected clones werescreened for expression by fractionation on SDS-PAGE gel and staining byCoommassie Blue and Western blot analysis using anti-HA andanti-flagellin antibodies.

Example 18 Generation of a Pam3Cys Fusion Protein

M2e (SEQ ID NO: 544) was chemically coupled to a tri-palmitoylcysteine(Pam3Cys) moiety through the amino terminal serine residue of thepeptide. The structure of the fusion protein (Pam3Cys.M2e) is shown inFIG. 39. The chemical name for Pam3Cys.M2e is[Palmitoyl-Cys((RS)-2,3-di(palmitoyloxy)-propyl)-Ser-Leu-Leu-Thr-Glu-Val-Glu-Thr-Pro-Ile-Arg-Asn-Glu-Trp-Gly-Ser-Arg-Ser-Asn-Asp-Ser-Ser-Asp-Pro-OHacetate salt]. The molecular mass of Pam3Cys.M2e is 3582.3 daltons.

Pam3Cys.M2e was synthesized using a solid phase peptide synthesismethodology based on a well established Fmoc-strategy (Houben-Weyl,2004. Synthesis of peptides and peptidomimetics, Vol. 22, Georg ThiemeVerlag Stuttgart, NY). The synthetic scheme and manufacturing processfor Pam3Cys.M2e is diagrammed in FIG. 81. The Pam3Cys.M2e is a fusionprotein (chemically linked) and is also referred to herein as a“lipidated peptide.”

The first step in the synthesis included solid phase peptide synthesis.The amino acid sequence of Pam3Cys.M2e was assembled on anH-Pro-2-chlorotrityl chloride resin by solid phase peptide synthesis.This resin is highly suitable for the formation of peptides with theFmoc-strategy. The peptide chain was elongated by successive coupling ofthe amino acid derivatives. Each coupling step was preceded by anFmoc-deprotection step and both steps were accompanied by repeatedwashing of the resin. After coupling of the last amino acid derivative,the final Fmoc-deprotection step was performed. Finally, the peptideresin was washed and dried under reduced pressure. During solid phasepeptide synthesis color indicator tests were performed for each step tomonitor the completion of the Fmoc-cleavage and the subsequent couplingof the amino acid derivatives.

Stage 2 of the synthesis included coupling of Pam3Cys-OH. Pam3Cys-OH waspre-activated with N,N′-dicyclohexyl-carbodiimide (DCCI) in the presenceof 1-hydroxybenzotriazole (HOBt). The resulting solution was filteredand added to the peptide resin. At the end of the reaction time thepeptide resin was washed and dried under reduced pressure. Colorindicator tests were performed to control the coupling of Pam3Cys-OH.

Stage 3 of the synthesis included cleavage from the resin includingcleavage of the side chain protecting groups. The peptide resin wastreated with trifluoroacetic acid (TFA). The product was precipitatedfrom the reaction mixture and lyophilized.

Stage 4 of the synthesis included purification by preparative reversephase HPLC. The crude material obtained from Stage 3 was purified bypreparative HPLC on a reverse phase column using a TFA system. Thefractions were collected, checked by analytical HPLC and pooledaccordingly. Pooled fractions from the TFA runs were lyophilized.

Stage 5 of the synthesis included precipitation in the presence of EDTA.The purified material from Stage 4 was precipitated from an aqueoussolution of EDTA. The product was filtered off and dried under reducedpressure.

Stage 6 of the synthesis included ion exchange chromatography. The laststage of manufacturing Pam3Cys.M2e was the exchange from thetrifluoroacetate salt into the acetate salt by ion exchange. Thematerial from Stage 5 was loaded onto an ion exchange column and elutedwith acetic acid. Fractions were checked by thin layer chromatographyand the combined product-containing fractions were filtered andlyophilized to yield the final product.

The purity specification for the Pam3Cys.M2e drug substance was ≧80% byRP-HPLC. The specification was based on the purity achieved with threenon-GMP lots of Pam3Cys.M2e made from the same GMP batch of M2e-peptideintermediate resin. The purity of the three non-GMP lots of Pam3Cys.M2ewas 80.2%, 80.3% and 80.8%, for lots D.001.Pam3Cys.M2e,D.002.Pam3Cys.M2e and D.003.Pam3Cys.M2e, respectively.

Example 19 Immunogenicity

Materials and Methods

Synthesis and Purification of Pam3Cys.M2E

Pam3Cys.M2e was prepared by Genemed Synthesis and Bachem using solidphase synthesis methodologies and FMOC chemistry as described above.Mass spectroscopy analysis was used to verify the molecular weight ofthe final product.

Endotoxin Assay

Endotoxin levels of the STF2.4×M2e and the Pam3Cys.M2e were measuredusing the QCL-1000 Quantitative Chromogenic LAL test kit (BioWhittaker#50-648U), following the manufacturer's instructions for the microplatemethod.

TLR5Bioactivity Assay

HEK293 cells constitutively express TLR5 and secrete several solublefactors, including IL-8, in response to TLR5 signaling. HEK293 cellswere seeded in 96-well microplates (50,000 cells/well) and test proteinswere added and incubated overnight. The next day, the conditioned mediumwas harvested, transferred to a clean 96-well microplate and frozen at−20° C. After thawing, the conditioned medium was assayed for thepresence of IL-8 in a sandwich ELISA using an anti-human IL-8 matchedantibody pair (Pierce, #M801E and #M802B) following the manufacturer'sinstructions. Optical density was measured using a microplatespectrophotometer (FARCyte, Amersham). Results are reported as pg of IL8per ml as determined by inclusion of a standard curve for IL8 in theassay.

TLR2Bioactivity Assay

RAW264.7 cells (ATCC) express TLR2 and secrete several soluble factors,including TNFα, in response to TLR2 signaling. RAW264.7 cells wereseeded in 96-well microplates (50,000 cells/well), test compounds wereadded and incubated overnight. The next day, the conditioned medium washarvested, transferred to a clean 96-well microplate and frozen at −20°C. After thawing, the conditioned medium was assayed for the presence ofTNFα in a sandwich ELISA using an anti-mouse TNFα matched antibody pair(Pierce) following the manufacturer's instructions. Optical density wasmeasured using a microplate spectrophotometer (FARCyte, Amersham).Results are reported as ng of TNF per ml as determined by reference to astandard curve for TNF included in the assay.

Mouse Immunogenicity

Female BALB/c mice (National Cancer Institute) were used at the age ofabout 6-8 weeks. Mice were divided into groups of 5 to 10 mice pergroup, and immunized subcutaneously on each side of the base of the tailon days 0 and 21 with the indicated concentrations of STF2.4×M2e orPam3Cys.M2e fusion protein. On days 10 (primary) and 28 (boost),individual mice were bled by retro-orbital puncture. Sera were harvestedby clotting and centrifugation of the heparin-free blood samples.

Mouse Serum Antibody Determination

M2e-specific IgG levels were determined by ELISA. 96-well ELISA plateswere coated overnight at 4° C. with 100 μl/well of a 5 μg/ml solution ofthe M2e peptide in PBS. Plates were blocked with 200 μl/well of AssayDiluent Buffer (ADB; BD Pharmingen) for one hour at room temperature.The plates were washed three times in PBS containing 0.05% Tween-20(PBS-T). Dilutions of the sera in ADB were added (100 μl/well) and theplates were incubated overnight at 4° C. The plates were washed threetimes with PBS-T. Horse radish peroxidase, or HRP-labeled goatanti-mouse IgG antibodies (Jackson Immunochemical) diluted in ADB wereadded (100 μl/well) and the plates were incubated at room temperaturefor 1 hour. The plates were washed three times with PBS-T. After addingTMB Ultra substrate (3,3′,5,5′-tetramentylbenzidine; Pierce) andmonitoring color development, the O.D. 450 was measured on a TecanFarcyte microspectrophotometer.

Rabbit Immunogenicity

Female and male NZW rabbits (Covance Research Products) were used at theage of about β-17 weeks. Rabbits were divided into groups of 3 male and3 female per group, and immunized i.m. on alternating thighs on days 0and 21 and 42 with the indicated concentrations of Pam3Cys.M2e peptideor STF2.4×M2e fusion protein. Animals were bled on day −1 (prebleed), 14(primary) and 28 and 42 (boost). Sera were prepared by clotting andcentrifugation of samples.

Rabbit Serum Antibody Determination

M2e-specific IgG levels were determined by ELISA. 96-well ELISA plateswere coated overnight at about 4° C. with 100 μl/well M2e peptide in PBS(5 μg/ml). Plates were blocked with 200 μl/well of Assay Diluent Buffer(ADB; BD Pharmingen) for one hour at room temperature. The plates werewashed three times in PBS-T. Dilutions of the sera in ADB were added(100 μl/well) and the plates were incubated overnight at about 4° C. Theplates were washed 3× with PBS-T. Bound IgG was detected usingHRP-conjugated goat anti-rabbit IgG (Jackson Immunochemical). The plateswere washed three times with PBS-T. After adding TMB Ultra substrate(Pierce) and monitoring color development, O.D. 450 was measured on aMolecular Devices Spectramax microspectrophotometer. Results arereported as the Delta O.D. which is determined by subtracting the O.D.450 reading for the prebleed of each animal from the O.D. 450 for eachanimal post-immunization.

BALB/C Mouse Efficacy Model

In a typical experiment, about 5-6 week old female BALB/c mice (10-20per group) were obtained and allowed to acclimate for one week. Fusionproteins formulated in PBS or other suitable formulation wereadministered by s.c. injection. Mice were immunized on days 0 and 14. Onday 21, sera was harvested by retro-orbital puncture and evaluated forM2e specific IgG by ELISA. Mice were challenged by intranasaladministration of 1×LD90 of the well characterized mouse adaptedInfluenza A strain, A/Puerto Rico/8/34 (H1N1). Mice were monitored dailyfor 14 days for survival and weight loss. Mice that lost about 30% oftheir initial body weight were humanely sacrificed, and the day ofsacrifice recorded as the day of death. Efficacy data were reported assurvival times.

Results

In Vitro Bioactivity

These assays were based on cell lines expressing the relevant TLR andscreened for the ability to produce either IL8 or TNF-α in response toTLR triggering. In FIG. 52, the ability of STF2.4×M2e (▪) or STF2.OVA(◯) to stimulate TLR5 dependent IL8 production was evaluated followingthe stimulation of TLR5 positive, HEK293 cells. The results indicatethat both fusion proteins stimulated IL8 production in a dose dependentmanner and that the activity of the PAMP was retained in the context ofthe fusion.

TLR2 activity was similarly evaluated for Pam3Cys.M2e followingstimulation of TLR2 positive RAW264.7□ cells. In FIG. 51, theexperimental groups are: the known endotoxin, LPS, as a positive control(♦), LPS plus the inhibitor of endotoxin polymixin B (PMB) as a negativecontrol (◯), free Pam3Cys as a positive control for TLR2 signalling (▪),free Pam3Cys plus PMB (□), Pam3Cys.M2e (♦) and Pam3Cys.M2e plus PMB (⋄).The results showed similar activity profiles for Pam3Cys.M2e and thefree TLR2 ligand Pam3Cys. The addition of polymyxin B (PMB) did notreduce its activity, indicating that there is no or low endotoxincontamination.

Physical Linkage of PAMP and Antigen Enhances Immunogenicity

Using mouse models of immunogenicity, chemical coupling of Pam3Cys toM2e enhances the immunogenicity of the M2e antigen as compared to eitherthe M2e peptide delivered alone or the M2e peptide co-delivered withfree Pam3Cys. In the experiment shown in FIG. 52, groups of mice wereimmunized on days 0 and 21 with PBS as a negative control (□*), the freeTLR2 ligand, Pam3CSK-4 (( ), M2e peptide alone (∘), free Pam3CSK-4 mixedwith M2e peptide (□), or the fusion of Pam3Cys and M2e referred to asPam3.M2e (♦). The relevant the molar ratio of M2e peptide delivered washeld constant. On day 28, sera were harvested and analyzed forM2e-specific antibody titers by ELISA. The results show that chemicalcoupling of Pam3Cys to the M2e (Pam3Cys.M2e) generates a detectableserum antibody response to the M2e antigen.

Physical linkage between the TLR5 ligand STF2 and antigen wasdemonstrated using the model antigen ovalbumin (OVA). Mice received asingle s.c. immunization with STF2, OVA, STF2.OVA fusion protein,STF2+OVA mixture or PBS alone. Dosages were calculated to deliver 12 μgequivalents of STF2 and OVA per group. Seven days later, sera wereharvested and OVA-specific antibodies were examined by ELISA. Data shownin FIG. 53 depict IgG1 titers at a 1:100 dilution of the sera. Theseresults demonstrate that physical linkage of the TLR5 ligand and antigenresults in optimal immunogenicity in vivo.

PAMP Linked Antigens are More Immunogenic than Conventional Adjuvant

Groups of 5 BALB/c mice were immunized on day 0 and 14 with 30 μg ofPam3Cys.M2e (♦), 22.5 μg of M2e which is the molar equivalent of M2e in30 μg of Pam3Cys.M2e (⋄), 22.5 mg of M2e adsorbed to the conventionaladjuvant Alum (□), or 25 mg of the recombinant protein STF2.4×M2e (▪). Agroup receiving PBS was included as a negative control (∘). Sera wereharvested 7 days post the second dose and M2e specific IgG wereevaluated by ELISA. The results shown in FIG. 54 indicate that M2e aloneis poorly immunogenic in that it failed to elicit antibody titers abovebackground. The conventional adjuvant Alum provided a modest enhancementin the immune response to M2e. The PAMP linked M2e constructs; however,provided the greatest enhancement in immunogenicity. These resultsindicate direct linkage of PAMPs with portions of an integral membraneprotein of an influenza viral protein can elicit immune responses thatare more potent than those elicited by the conventional adjuvant Alum.

Dose and Immunogenicity

Dose ranging studies were carried out to further assess the potency ofPam3Cys.M2e and STF2.4×M2e. For STF2.4×M2e, BALB/c mice were immunizedon day 0 and 14 with dilutions of STF2.4×M2e that ranged from 0.25 to 25μg of STF2.4×M2e per immunization. The prefix D002 refers to thespecific batch of STF2.4×M2e used in this experiment, while R-028 refersto a historical reference batch of STF2.4×M2e used in this experiment.Seven days following the last immunization (Day 21) mice were bled andM2e-specific IgG responses were evaluated by ELISA. The results shown inFIG. 55 demonstrate that immunization with doses as low as 0.25 μg perimmunization of STF2.4×M2e induced detectable levels of M2e-specificIgG, with the optimal dose in mice falling in the range of about 2.5 toabout 25 μg.

For Pam3Cys.M2e, BALB/c mice were immunized on day 0 and 14 with 0.05 to30 μg of Pam3Cys.M2e per immunization. Seven days following the lastimmunization (Day 21) mice were bled and M2e-specific IgG responses wereevaluated by ELISA. The results shown in FIG. 56 demonstrate thatimmunization with concentrations as low as 0.05 μg of Pam3Cys.M2einduced detectable levels of M2e-specific IgG, with the optimal dose formice in this study of about 30 μg.

Immunogenicity in Multiple Mouse Strains

The immunogenicity of Pam3Cys.M2e was evaluated in multiple mousestrains including BALB/c (●), C57BL/6 (▪), CB6/F1 (♦), DBA/2 (▴), Cr:NIH(Swiss) (X) and C3H/HeN (*). Groups of five for each strain wereimmunized on day 0 and 14 with 30 μg of Pam3Cys.M2e per immunization.Sera were harvested on day 21 and levels of M2e-specific IgG evaluatedby ELISA. All strains exhibited significant levels of M2e-specific IgGindicating that the immunogenicity of Pam3Cys.M2e is not dependent on aparticular MHC (FIG. 57).

Immunogenicity in Rabbits

Studies aimed at evaluating the immunogenicity of Pam3Cys.M2e andSTF2.4×M2e in a second species, rabbit, were carried out. In the firststudy, rabbits (3 females and 3 males/group) were immunized with 500,150, 50, 15 or 5 μg (i.m.) of Pam3Cys.M2e on day 0, 21 and 42. As acontrol, an additional group received the formulation buffer F111 (10 mMTris, 10 mM histidine, 75 mM NaCl, 5% sucrose, 0.02% Polysorbate-80, 0.1mM EDTA, 0.5% ethanol, 20 mg/mL hydroxypropyl-beta-cyclodextrin, pH7.2). On day 7 post-boost 2, peripheral blood was obtained and theanti-M2e antibody titers were evaluated by ELISA. The results shown inFIG. 58 depict the individual rabbit antibody titers at a 1:125 dilutionof the sera. The data suggest a dose-response relationship between theamount of Pam3Cys.M2e used for prime/boost vaccinations and the level ofthe antibody titer achieved.

In the second study, rabbits (3 females and 3 males/group) wereimmunized with 500, 150, 50, 15 or 5 μg (i.m.) of STF2.4×M2e. As acontrol, an additional group received saline alone. On day 14post-immunization, peripheral blood was obtained and the anti-M2eantibody titers were evaluated by ELISA. Notably, significantM2e-specific IgG responses were detectable by day 14 post-prime in allanimals immunized (FIG. 59). The results indicate that STF2.4×M2eelicits a rapid and consistent immune response in rabbits.

Efficacy in the Mouse Challenge Model

The efficacy of the Pam3Cys.M2e and STF2.4×M2e was evaluated in BALB/cmice using the well characterized mouse adapted strain, InfluenzaA/Puerto Rico/8/34 (PR/8) as the challenge virus. Groups of ten micewere immunized s.c. on day 0 and 14 with 30 μg of Pam3Cys.M2e in theformulation buffer F111 (▪), 30 μg of Pam3Cys.M2e in the proprietarybuffer F120 (10 mM Tris, 10 mM histidine, 10% sucrose, 0.02%Polysorbate-80, 0.1 mM EDTA, 0.5% ethanol, 0.075% docusate sodium, pH7.2) (▴), 30 μg of Pam3Cys.M2e in the buffer F119 (10 mM Tris, 10 mMhistidine, 75 mM NaCl, 5% sucrose, 0.02% Polysorbate-80, 0.1 mM EDTA,0.5% ethanol, 0.1% docusate sodium, pH 7.2), 30 μg of STF2.4×M2e in thebuffer F105 (10 mM Tris, 10 mM histidine, 75 mM NaCl, 5% sucrose, 0.02%Polysorbate-80, 0.1 mM EDTA, 0.5% ethanol, pH 7.2), 3 μg of STF2.4×M2ein buffer F105 (10 mM Tris, 10 mM histidine, 75 mM NaCl, 5% sucrose,0.02% Polysorbate-80, 0.1 mM EDTA, 0.5% ethanol, pH 7.2) (•) or 0.3 μgof STF2.4×M2e in buffer F105 (□). A group receiving PBS alone wasincluded as a negative control (∘), and a convalescent group withimmunity to PR/8 following a sublethal challenge with the virus wasincluded as a positive control (⋄). On day 28, animals were challengewith an LD90 of the PR/8 challenge stock. Weight loss and survival wasfollowed for 14 days post challenge (FIG. 60).

Animals in the convalescent group which had successfully cleared anearlier non-lethal infection with PR/8 demonstrated 100% protection to asubsequent viral challenge. Animals receiving the PBS buffer aloneexhibited morbidity beginning on days 7 and 8, with 80% lethalityoccurring by day 10, while animals immunized with 30 μg of Pam3Cys.M2ein F111 demonstrated enhanced survival, with 50% of mice surviving thechallenge. Animals receiving Pam3Cys.M2e in F119 exhibited morbiditybeginning on days 8 and 9 with 80% of the mice surviving. Animalsreceiving Pam3Cys.M2e in buffer F120 (10 mM Tris, 10 mM histidine, 10%sucrose, 0.02% Polysorbate-80, 0.1 mM EDTA, 0.5% ethanol, 0.075%docusate sodium, pH 7.2) or the STF2.4×M2e protein exhibited the mildestdisease course with 90 to 100% of the mice in these groups surviving thelethal challenge. These results demonstrate that both Pam3Cys.M2e andSTF2.4×M2e can confer protective immunity to a challenge with influenzaA in vivo.

Discussion

Salmonella typhimurium flagellin (fljB) is a ligand for TLR5. Arecombinant protein consisting of full-length flijB (STF2) fused to fourtandem repeats of M2e was expressed in E. coli and purified to >95%purity with low endotoxin levels. In reporter cell lines, this protein(STF2.4×M2e) triggered IL8 production in a TLR5-dependent fashion. Miceimmunized with dilutions of STF2.4×M2e that ranged from 0.25 μg to 25μg, formulated in the buffer F105 which is without a conventionaladjuvant or carrier, mounted a vigorous antibody response. The potencyof the recombinant protein was further demonstrated in rabbitimmunogenicity studies where animals receiving as little as 5 μg ofprotein seroconverted after a single dose. The efficacy of the PAMPfusion protein was demonstrated in the mouse challenge model usingInfluenza A/Puerto Rico/8/34 as the challenge virus. Mice immunized withas little as about 0.3 μg of the protein per dose exhibited mildmorbidity with 100% of the mice surviving the challenge.

Synthetic tripalmitoylated peptides mimic the acylated amino terminus oflipidated bacterial proteins and are potent activators of TLR2. In thesestudies, a tripalmitoylated peptide consisting of three fatty acidchains linked to a cysteine residue and the amino terminus of theInfluenza A M2 ectodomain (M2e) was synthesized using standardsolid-phase peptide chemistries. This peptide (Pam3Cys.M2e) triggeredTNFα production in a TLR2-dependent fashion in reporter cell lines. Whenused to immunize mice without adjuvant, Pam3Cys.M2e generated anantibody response that was more potent than M2e when mixed with freePam3CSK-4. Pam3Cys.M2e was also found to be immunogenic in rabbits wherea dose response relationship was observed between the amount ofPam3Cys.M2e used for immunization and the antibody titer achieved. Theefficacy of the Pam3Cys.M2e peptide in a number of differentformulations was evaluated in the mouse challenge model using InfluenzaA/Puerto Rico/8/34 as the challenge virus. Pam3Cys.M2e formulated inF119 and F120 exhibited the mildest morbidity with about 80 to about100% of the mice surviving the challenge.

Example 20 Materials and Methods

PCR Amplification and DNA Primers

All PCR amplifications were performed using Pfu Ultra Hotstart PCRMaster Mix (Catalog number 600630) from Stratagene (La Jolla, Calif.)according to the manufacturer's recommendations. DNA primers werepurchased from Sigma Genosys and are described below.

STF28BGF-1: (SEQ ID NO: 644) CTCGGGAGATCTGCACAAGTAATCAACACTAACAGTCTSTF28MCR-1: (SEQ ID NO: 645) CCATGGGCTAGCAGGATCCACCGGCGCTCCCTGCACGTTCASTF28MCF-2: (SEQ ID NO: 646) GGAGCGCCGGTGGATCCTGCTAGCCCATGGACCGAAAACCCGSTF28ECR-2: (SEQ ID NO: 647)TCTGCAGAATTCACGTAACAGAGACAGCACGTTCTGCGGGACGTCCCGCAGAACGTGCTGTCTCTGTTACGTGAATTCTGCAGA pET24AR:5 (SEQ ID NO: 648)TCCGGCGTAGAGGATCGAGA STF2-E3R3: (SEQ ID NO: 649)CAATTGACCTTCAAGCTTCGAATTGCCCTTACGTAACAGAGACAGCACGT TCTG AX-E3F3: (SEQ IDNO: 650) AAGCTTGAAGGTCAATTGGAATTCCCTAGGACTAGTATGGAAAAATTGC AGTTGAAGpET24AF: (SEQ ID NO: 651) GCTTAATGCGCCGCTACAGG 5′WNE28: (SEQ ID NO: 652)GCGGCCGCTCATGGAAAAATTGCAGTTGAAGGGAACAACC 3′WNE28: (SEQ ID NO: 653)CCGCGGTTTGCCAATGCTGCTTCCAGACTTGT NdeI-STF2: (SEQ ID NO: 654)CCGGCATGCCATATGGCACAAGTAATCAACACTAACAGTCTGTCGCTGC BlpI-EdIII: (SEQ IDNO: 656) GCATGCTCAGCTTATTAAGGGTTTGCCAATGCTGCTTCCCAGACTTGTG JE EIIIprimer: (SEQ ID NO: 53) TACGTGAATTCAGCAGATATCCAGCACCloning of pET/STF2Δ.EIII

Full length flagellin of Salmonella typhimurium fljb (flagellin phase 2)(also referred to herein as “STF2”) is encoded by a 1.5 kb gene. Atruncated version of the STF2 (STF2Δ, SEQ ID NO: 606, encoded by SEQ IDNO: 607) was generated by deleting the hyper-variable region that spansamino acids 170 to 415 of SEQ ID NO: 604. The deleted region wasreplaced with a short flexible linker (GAPVDPASPW, SEQ ID NO: 659)designed to facilitate interactions of the NH2 and COOH terminisequences necessary for TLR5 signaling. To generate this construct, atwo-step PCR was used. In the first reaction, STF2.OVA (SEQ ID NO: 755encoding amino acid sequence SEQ ID NO: 756) served as the DNA templateand STF28BGF-1 and STF28MCR-1 were used as primer pairs. In a separatereaction, the same DNA template was combined with primers STF28MCF-2 andSTF28ECR-2.

The PCR amplification reactions generated about 500 bp and about 270 bpfragments, respectively. These PCR products were combined in a final PCRreaction using STF28BGF-1 and STF28ECR-2 as primers. The amplified DNAproduct from this reaction (about 0.77 kb) was digested with BglII andEcoRI restriction enzymes and ligated into pMTBiP/V5-His B (Invitrogen,Carlsbad, Calif.) that had previously been digested with BglII and EcoRIand treated with alkaline phosphatase. An aliquot of the ligation mixwas used to transform TOP10 cells (InVitrogen, Carlsbad, Calif.). PCRscreening was performed using vector specific primers, pMTFOR(methionine promoter) (CATCTCAGTGCAACTAAA, SEQ ID NO: 759) and BGHREV(bovine growth hormone poly A) (TAGAAGGCACAGTCGAGG, SEQ ID NO: 760), toidentify several positive clones. All positive clones were furtheranalyzed by restriction mapping analysis and confirmed by DNAsequencing. The resultant construct pMT/STF2Δ was used to generatepMT/STF2Δ.EIII+.

The domain III of the West Nile virus envelope protein ofpET/STF2Δ.EIII+ (SEQ ID NOS: 673, 674) was derived from the Drosophilaexpression plasmid pMT/STF2.E. This plasmid contains full-length STF2(amino acids 1-506, SEQ ID NO: 604) fused to the West Nile Virusenvelope protein (amino acids 1-406, SEQ ID NO: 642). The pMT/STF2.E(SEQ ID NO: 761) clone AX-1 was used as a DNA template and 5′WNE28 (SEQID NO: 652) and 3′WNE28 (SEQ ID NO: 653) served as primers for PCRamplification. In order to facilitate restriction analysis andsubsequent cloning steps, the 5′ primer encoded a novel NotI site (NewEngland Biolabs, Beverly, Mass.) and the 3′ primer contained a uniqueSacII site. The amplified EIII+ DNA fragment (345 bp; SEQ ID NO: 781that encodes amino acids 292-406 of SEQ ID NO: 642) was subcloned intopCR-Blunt II-TOPO cloning vector (InVitrogen, Carlsbad, Calif.) togenerate plasmid TOPOEIII. A stop codon was subsequently introduceddownstream of the EIII+ sequence by blunting the SacII and SpeIrestriction sites using T4 DNA polymerase.

To generate pMT/STF2Δ.EIII+ (SEQ ID NOS: 673, 674), the EIII+ fragmentwas isolated from TOPOEIII+ using NotI and BamHI restriction sites andligated into the NotI and SacII restriction sites in pMT/STF2Δ. TheBamHI site of the EIII+DNA fragment and the SacII site of pMTSTF2Δ wereblunted with T4 DNA polymerase prior to ligation. The STF2Δ.EIII+sequence (SEQ ID NOS: 673, 674) from pMT/STF2Δ.EIII+ was isolated by PCRamplification using the primers NdeI-STF2 and BlpI-EdIII. To generatepET/STF2Δ.EIII+ (SEQ ID NO: 674), the PCR product was digested with NdeIand BlpI and ligated into pET24a plasmid that had been predigested withNdeI and BlpI. The ligation mix was transformed into Mach-1 cells(InVitrogen, Carlsbad, Calif.) and the cells were grown on LBsupplemented with 50 μg/ml kanamycin. Several colonies were screened byrestriction mapping and were verified by DNA sequencing.

Cloning of pET/STF2.EIII+

The West Nile virus EIII+ sequence of pET/STF2.EIII+ (SEQ ID NOS: 657,658) was derived from pETSTF2.E (SEQ ID NOS: 761, 762). This E. coliexpression plasmid contains full-length STF2 (amino acids 1-506) fusedto the West Nile Virus envelope protein (amino acids 292-406 of SEQ IDNO: 642, which is SEQ ID NO: 610). In two independent PCR reactions,pET/STF2.E was used as the DNA template. One reaction used the primerspET24AR:5 (SEQ ID NO: 648) and STF2-E3R3: (SEQ ID NO: 649) and the otherused AX-E3F3 (SEQ ID NO: 650) and pET24AF (SEQ ID NO: 651). These PCRreactions generated a 1.5 kb fragment that consisted of full-length STF2and a 340 bp fragment that comprised the EIII domain plus additionalamino acids that extended into domain I of the envelope protein.Aliquots of these PCR amplification reactions were combined, and the twoproducts served as templates for a PCR reaction with the externalprimers pET24AR (SEQ ID NO: 648) and pET24AF (SEQ ID NO: 651). Thisresulted in the generation of about a 1.8 kb DNA fragment that fusedEIII+ sequence (SEQ ID NO: 781, a nucleic acid sequence encoding aminoacids 292-406 of SEQ ID NO: 642, which is SEQ ID NO: 610) to STF2. ThePCR product was digested with NdeI and BlpI and gel purified and ligatedby compatible ends to a pET24a vector that had previously been digestedwith compatible enzymes and de-phosphorylated. The ligation mix wastransformed into Mach-1 cells (InVitrogen, Carlsbad, Calif.) asdescribed for pET/STF2Δ.EIII+. Several colonies were screened byrestriction mapping and two clones were verified by DNA sequencing.

Cloning of pET/STF2Δ.JEIII+

A portion of the envelope protein of a Japanese encephalitis virus (JEV)(strain SA-14-14-2 (Jai, L., et al., Chin Med J (Eng) 116:941-943(2003)); currently employed in a JEV vaccine encoded by domain III wascustom synthesized by DNA 2. Inc (Menlo Park, Calif.). The portion ofdomain III was excised from the pJ2:G01510 using NotI and Blp I sitethat flank the insert. The DNA insert was gel isolated and cloned bycompatible ends to pET24A/STF2Δ.EIII+ (SEQ ID NOS: 673, 674) that hadpreviously been digested with the appropriate enzymes to release theWest Nile virus EIII+ insert. The deleted vector was then gel purifiedand ligated to an aliquot of JE EIII+. The ligation mix was used totransform TOP-10 cells (InVitrogen, Carlsbad, Calif.) and the cells weregrown on LB supplemented with 50 μg/ml kanamycin. Several colonies werescreened by restriction mapping and were verified by DNA sequencing.

The resulting construct, pET24A/STF2Δ.JEIII (SEQ ID NOS: 608, 609) wasBLR (DE3) strain (Novagen) and expression was monitored in severalclones using Commassie Blue staining which was confirmed by Western blotusing anti-flagellin antibodies. Using, pET24A/STF2Δ.JEIII+ as the DNAtemplate and the JE EIII+ oligonucleotide as primer (SEQ ID NO: 656) thecysteine residue in the linker region between STF2Δ and JEIII+ waschanged to a serine residue using QuikChange Site Directed MutagenesisKit (Stratagene, La Jolla, Calif.) according to the manufacturer'sinstructions. The clone was verified by sequencing and assayed forexpression as described for pET24A/STF2Δ.JEIII+ above.

When a cysteine residue in a linker in change to a serine residue thefusion protein in also referred to herein by inclusion of an “s” in thedesignation of the fusion protein. For example, “STF2Δ.EIII+” includes acysteine residue in the linker (SEQ ID NO: 674), whereas “STF2Δ.EIIIs+”include a serine residue substituted for the cysteine residue in thelinker (SEQ ID NO: 675).

Cloning the EIII Domain of Each Dengue Virus Fused to the C-Terminal Endof Flagellin (STF2Δ)

Initially, obtaining biologically active material from the fusion of theentire envelope protein of West Nile virus was difficult, perhaps due tothe presence of multiple cysteines residues (12 cysteines) in theenvelope protein (see SEQ ID NO: 642). However, when the region encodingdomain III (EIII) of the protein was sub-cloned, the fusion protein wasabundantly expressed in E. coli and was highly efficacious in mice.Although there is an overall sequence dissimilarity among the 4 distinctDEN viruses (Den1, Den2, Den3, Den4, SEQ ID NOS: 763-770, thethree-dimensional structures within domain III of the envelope proteinare similar among the flaviviruses. This domain in DEN and otherflaviviruses encodes the majority of the type-specific contiguouscritical/dominant neutralizing epitopes. Domain III of the dengueviruses (Den1, Den2, Den3 and Den4) has been expressed in bacteria andshown to be immunogenic, capable of inducing neutralizing antibodies inexperimental animals (Simmons, M., et al., Am. J. Trop. Med Hyg 65:159(2001)). Domain III corresponding to residues about 295 to about 399(exact numbering depends on the particular DEN virus, for example, ofSEQ ID NOS: 763, 765, 767, 769) of the four different DEN viruses havebeen codon-optimized for expression in E. coli. The synthetic gene wasamplified by using PCR and sub-cloned into the NotI site of the vectorpET/STF2Δ generating pET/STF2Δ.DEN1EIII, pET/STF2Δ.DEN2EIII,pET/STF2Δ.DEN3EIII and pET/STF2Δ.DEN4EIII (SEQ ID NOS: 683, 685, 687 and689).

E. coli Production of STF2.EIII+, STF2Δ.EIII+, STF2Δ.EIIIs+ andSTF2Δ.JEIII+

Cell cultures (6 L) of BLR(DE3) pLysS that harbor pETSTF2.EIII+ (SEQ IDNOS: 657, 658), pETSTF2Δ.EIII+ (SEQ ID NOS: 673, 674), pETSTF2Δ.EIIIs+(SEQ ID NOS: 675, 676) or pETSTF2Δ.JEIII+SEQ ID NOS: 608, 609) weregrown in LB medium containing 15 μg/ml kanamycin, 12.5 μg/mltetracycline and 24 μg/ml chloramphenicol. At an OD₆₀₀ of about 0.6protein expression was induced with 1 mM IPTG for about 3 h at about 37°C. Following induction, cells were harvested by centrifugation (7000rpm×7 minutes in a Sorvall RC5C centrifuge) and resuspended in 2×PBS, 1%glycerol, DNAse, 1 mM PMSF, protease inhibitor cocktail and 1 mg/mllysozyme. The suspension was passed through a microfluidizer to lyse thecells and the lysate was centrifuged (45,000 g for one hour in a BeckmanOptima L ultracentrifuge) to separate the soluble fraction frominclusion bodies. Under these growth and induction conditions,STF2.EIII+ was expressed as a soluble protein and STF2Δ.EIII+(SEQ IDNOS: 673, 674), STF2Δ.EIIIs+ (SEQ ID NOS: 675, 676) and STF2Δ.JEIII+(SEQ ID NOS: 608, 609) formed inclusion bodies.

Purification of STF2.EIII+

Cell lysate containing soluble STF2.EIII+ (SEQ ID NOS: 657, 658) wasapplied to Sepharose Q resin (Amersham Biosciences, Piscataway, N.J.) inthe presence of 0.5 M NaCl to reduce DNA, endotoxin, and othercontaminants. The flow-through fraction was collected and theconductivity adjusted by a 10-fold dilution with buffer A (Buffer A: 100mM Tris-C1, pH 8.0). The diluted material was re-loaded onto Q Sepharoseand bound protein was eluted with a linear gradient from 20% to 60%Buffer B (Buffer B: 100 mM Tris-C1, 1 M NaCl, pH 8.0). Fractionscontaining STF2.EIII+ were pooled and further processed by Superdex-200gel (SD200) filtration chromatography in the presence of Na-deoxycholateto remove residual endotoxin (running buffer: 1% Na-deoxycholate, 100 mMNaCl, 100 mM Tris-HCl, 1% glycerol, pH 8.0). Following SD200chromatography, the eluted protein was loaded directly onto Q Sepharoseand washed extensively with buffer A to remove detergent. Bound proteinwas again eluted with a linear gradient from 20% to 60% Buffer B. In onepreparation (Batch 057), this step was substituted with a detergentremoval procedure using Extract-D detergent removal gel (PierceBiotechnology, Rockford, Ill.). The purified protein was dialyzedagainst buffer containing 50 mM Tris, 100 mM NaCl and 1% glycerol andstored at −80° C.

Purification of STF2Δ.EIII+

STF2Δ.EIII+ inclusion bodies were collected by low-speed centrifugation(7000 rpm×7 minutes in a Sorvall RC5C centrifuge) and solubilized withbuffer containing 8 M urea, 100 mM Tris-HCl, 5 mM EDTA, pH 8.0. The ureaconcentration of the solubilized protein was adjusted to 1 M and thesample was loaded onto Q Sepharose. The bound protein was eluted using alinear gradient from 0% to 100% Buffer B. (Buffer A: 100 mM Tris-HCl, 5mM EDTA, 1 M urea, pH 8.0. Buffer B: 100 mM Tris-Cl, 5 mM EDTA, 1 MNaCl, 1 M urea, pH 8.0). Due to the formation of protein aggregatesfollowing elution, the urea concentration of the Q Sepharose materialwas adjusted to 8 M. The protein was further purified by gel filtrationchromatography using SD200. The column was pre-equilibrated with 100 mMTris-HCl, pH 8.0, 100 mM NaCl, 1% glycerol, 8 M urea plus 1%Na-deoxycholate. The eluted protein was subjected to a second IEXchromatography step using Source Q to remove 1% Na-deoxycholate. Boundprotein was eluted with a linear gradient from 20% to 60% Buffer B.(Buffer A: 100 mM Tris-C1, pH 8.0, 8 M urea, 5 mM EDTA. Buffer B: 100 mMTris-HCl, pH 8.0, 5 mM EDTA, 8 M urea, 1 M NaCl). Final polishing of theprotein was completed by gel filtration chromatography using SD200(Running Buffer: 100 mM Tris-HCl, pH 8.0, 8 M urea, 100 mM NaCl and 1%glycerol). Reducing agent was added to the SD200 fraction (2.5 mM DTT)and the protein was refolded by step-wise dialysis against decreasingconcentrations of urea. The urea concentration was reduced sequentiallyagainst buffers that contained 100 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1%glycerol and 6 M, 4 M, 2 M or no urea.

Refolding and Purification of STF2Δ.EIII+ Trimer

STF2Δ.EIII+ (SEQ ID NOS: 673, 674) from urea-solubilized inclusionbodies was efficiently refolded to form trimer product by simpledialysis as described above the trimer (3 of thes STFΔ.EIII fusionproteins) was deduced based on molecular weight in SDS-PAGE. Followingdialysis, endotoxin was removed by multiple extractions with TritonX-114. The trimer was purified and separated from monomer and aggregatesby S200 size exclusion chromatography. The final product migrated as asingle band with an apparent molecular weight of about 130 kDa onSDS-PAGE.

Refolding and Purification of STF2Δ.EIII+ Monomer

The monomeric form of STF2Δ.EIII+ (SEQ ID NOS: 673, 674) was producedconsistently and efficiently by refolding using rapid dilution, whichprevented individual STF2Δ.EIII+ fusion proteins from interacting withone another to form meutimers, such as trimers (supra). STF2Δ.EIII+solubilized from inclusion bodies in 4M urea was raised to 8M ureawithout reductant. The protein was then rapidly diluted inTris/NaCl/glycerol buffer, pH 8.0, to about 0.1 mg/ml and a final ureaconcentration of 0.1M at room temperature. The monomer was furtherpurified and separated from aggregates by 5200 size exclusionchromatography. The final product migrated as a single band with anapparent molecular weight of about 43 kDa on SDS-PAGE.

Purification of STF2Δ.EIIIs+ (Serine Substitution of the Linker BetweenSTF2Δ and EIII+, SEQ ID NO: 675)

STF2Δ.EIIIs+ (SEQ ID NOS: 675, 676) from solubilized inclusion bodieswas refolded using a rapid dilution method similar to that used torefold the STF2Δ.EIII+ monomer. The refolded protein was captured on abutyl sepharose column and eluted while removing most of the endotoxincontamination. Eluate from the butyl sepharose purification wasconcentrated and put through 4 cycles of Triton X-114 extractions toreduce endotoxin levels down to about <0.1 EU/μg before a finalpurification step over SD200 gel filtration. The final pooled productmigrated as a single band with an apparent molecular weight of about 43kDa on SDS-PAGE and contained a trace amount of Triton X-114 (about0.000015%).

Purification of STF2Δ.JEIII+ (SEQ ID NOS: 608, 609)

Protein was isolated from inclusion bodies under denaturing conditions.Inclusion bodies were washed with detergent (0.5% Triton X 100) andsolubilized in 8 M urea, resulting in partial purification of the targetprotein. For endotoxin removal, protein was applied on a Source S cationexchange column at low pH (about 3.5) and eluted with a salt gradient (0to about 1M NaCl). The protein was refolded using rapid dilution asdescribed for STF2Δ.EIII+ monomer. The protein was then concentrated andfurther purified using SD200 to separate the monomeric form of theprotein from aggregates. The purified material migrated with an apparentmolecular weight of about 43 kDa on SDS PAGE and contained acceptablelevels of endotoxin (about 0.03 EU/ug).

Fed Batch Production of Fusion Proteins

STF2Δ.EIIIs+ was produced in an aerobic bioreactor using a fed batchprocess. Three control loops were placed to control pH by acid (2 N HCl)or base (3 N NH₄OH) addition, temperature by heating (heating blanket)or cooling (time cycled cooling loop), and dissolved oxygen bycompressed air flow (manually controlled), agitation (mixing speed) andO₂ flow (timed cycled) in cascade. Cells [BLR(DE3) pLysS that harbor theSTF2Δ.EIIIs+ were adapted to and banked in MRSF media (see infra), andfrozen in 25% glycerol. Cells were scaled up for the bioreactor byadding 1 mL of banked cells to 1 L of MRSF media and agitating at about37° C. for about 15.5 to about 16.5 hours. Cells from the scale upprocess were added in a about 1:10 ratio to MRSF or MRBR synthetic mediaat about 37° C. and about 0.5 vvm air flow.

The process was run in batch mode at about 37° C. until the cells oxygenconsumption was such that the compressed air flow is about 1.5 vvm andthe agitation is at the maximum, about 6 hours, when the temperature isdropped to between about 25° C. and about 33° C. The feed can be startedbefore the culture is induced, or up to about 1 and about ½ hours after.The feed rate can be kept constant, or adjusted based on processvariables (dissolved oxygen, glucose concentration). The culture wasinduced with IPTG upon batch glucose exhaustion. The culture wasmaintained for a minimum of about 2 hours and a maximum of about 20hours.

MRBR Media Composition g/L Glucose 20 KH₂PO₄ 2.2 (NH₄)₂SO₄ 4.5 CitricAcid 1.0 MgSO₄(7H₂0) 1.0 CaCl₂ 0.04 Trace Metals 1 ml Thiamine HCl 0.01Antifoam 0.05

MRSF Media Composition g/L Glucose 10 (20 in bioreactor) KH₂PO₄ 7.8(NH₄)₂SO4 2.33 Citric Acid 1.0 MgSO₄(7H₂0) 1.0 CaCl₂ 0.04 Trace Metals 1ml Thiamine HCl 0.01 Kanamycin 0.0075 (shake flask only)

Trace Metal Solution 1000x Component g/L EDTA, disodium 5 FeSO₄(7H₂O) 10ZnSO₄(7H₂O) 2 MnSO₄(H₂O) 2 CoC1₂(6H₂O) 0.2 CuSO₄(5H₂O) 0.1 Na₂MoO₄(2H₂O)0.2 H₃BO₃ 0.1

Feed Media Composition g/L NaC1 0.5 FeSO₄(7H₂O) 2 CaC1₂ 3.5 MgSO₄(7H₂O)12 Thiamine HC1 1 Trace Metals 1 ml Glucose 100

STF2Δ.EIIIs+ was produced as inclusion bodies. Upon harvest, the cellswere separated from the conditioned media by centrifugation (BeckmanAvanti J-20 XP, JLA 8.1000 rotor, 10 k×g for about 20 minutes at about4° C.) and resuspended in equal volume of 50 mM Tris, 100 mM NaCl, 1 mMEDTA, pH 8.0. The centrifugation was repeated under the same conditions,with the cells resuspended in a minimum volume of the same buffer. Thesuspension was passed through a homogenizer (APV-1000) at >10,000 psifor at least two passes.

The solids can be separated and the STF2Δ.EIIIs solubilized by one ofthree methods; centrifugation, filtration, or fluidized bedchromatography.

Method 1

Solids are separated by centrifugation (Beckman Avanti J-20 XP, JA 20rotor, 20 k×g for 20 minutes at 4° C.) and resuspended in 50 mM tris, 1m NaCl, 1 mM EDTA, 1% glycerol, 0.5% Triton X-100, pH 8.0. This processwas repeated up to 6 times (total) at increasing speeds and times (to amaximum of about 40 k×g for about 20 minutes). After the final pelletrecovery, the pellet was resuspended in 50 mM Tris, 0.1M NaCl, 1 mMEDTA, pH 8.0 and clarified by centrifugation (Beckman Avanti J-20 XP, JA20 rotor, 40 k×g for about 20 minutes at about 4° C.) The pellet wasresuspended and dissolved in 50 mM Tris, 0.1M NaCl, 1 mM EDTA, 4 M urea,pH 8.0. Insolubles were removed by centrifugation (Beckman Avanti J-20XP, JA 20 rotor, 40 k×g for about 50 minutes at about 4° C.), thesupernatant retained for further processing.

After the multiple washes described above, STF2Δ.EIIIs can also bedissolved in 50 mM acetate, 10 mM NaCl, 8M urea, pH about 4.1 to about5.3 and clarified by centrifugation (Beckman Avanti J-20 XP, JA 20rotor, 20 k×g for about 20 minutes).

Method 2

After homogenization, the lysate was captured in body feed andSTF2Δ.EIIIs+ extracted with urea containing buffer. Body feed is afilter aid designed to trap particles in a cake above a depth filter.The body feed (Advanced Minerals Corporation CelPure 65) is a diatomite(silica powder) with a high surface area and low permeability, retaining<0.2 μm particles. The filter aid was pre-mixed with the lysate andpumped over a depth filter (Ertel 703), building up a cake containingboth body feed and lysate particles. The suspension creates a depthfilter as the particles settle on the filter pad. A 50 mM Tris, 100 mMNaCl pH 8.0 wash was performed to remove soluble proteins and nucleicacids. A subsequent wash with 50 mM Tris, 100 mM NaCl, 4 M urea, pH 8solubilizes and removes the STF2Δ.EIIIs from the body feed for furtherprocessing.

Method 3

After the cells were initially resuspended in buffer, they wereresuspended in sodium chloride and urea containing buffer at pH about 6to about 8 and homogenized. The lysate was applied on a Streamline CSTfluidized bed column (GE Healthcare) where the STF2Δ.EIIIs+ binds to theresin and the particulates flow through. STF2Δ.EIIIs+ may be eluted inlow salt conditions at a pH greater than the load pH, in the presence orabsence of detergents such as Triton X-100 or polysorbate 80.

SDS-PAGE

Proteins (typically about 5 μg) were diluted in SDS-PAGE sample bufferwith and without β-mercaptoethanol. The samples were boiled for 5minutes and loaded onto a 4-20% SDS polyacrylamide gel. Followingelectrophoresis, gels were stained with coomassie blue to visualizeprotein bands.

Endotoxin Assay

Endotoxin levels were measured using the QCL-1000 QuantitativeChromogenic LAL test kit (BioWhittaker #50-648U, Walkersville, Md.),following the manufacturer's instructions for the microplate method.

Protein Assay

Protein concentrations were determined by the MicroBCA Protein AssayReagent Kit in a 96-well format using BSA as a standard (PierceBiotechnology, Rockford, Ill.).

TLR5 Bioactivity Assay

HEK293 cells (ATCC, Catalog No. CRL-1573 Manassas, Va.) constitutivelyexpress TLR5, and secrete several soluble factors, including IL-8, inresponse to TLR5 signaling. Cells were seeded in 96-well microplates(about 50,000 cells/well), fusion proteins added and incubatedovernight. The next day, the conditioned medium was harvested,transferred to a clean 96-well microplate, and frozen at −20° C. Afterthawing, the conditioned medium was assayed for the presence of IL-8 ina sandwich ELISA using an anti-human IL-8 matched antibody pair (Pierce,#M801E and #M802B, Rockford, Ill.) following the manufacturer'sinstructions. Optical density was measured using a microplatespectrophotometer (FARCyte, Amersham Biosciences, Piscataway, N.J.).

Plaque Reduction Neutralization Test (PRNT)

PRNT was performed according to Wang, et al., J. Immunol. 167:5273-5277(2001). Briefly, serum samples were heat inactivated by incubation in a56° C. water bath for about 30 min and were serially diluted in PBS with5% gelatin from 1/10 to 1/2560. West Nile virus was diluted in PBS with5% gelatin so that the final concentration was about 100 PFU/well. Viruswas mixed with about 75 μl serum in a 96-well plate at about 37° C. forabout 1 h. Aliquots of serum-virus mixture were inoculated ontoconfluent monolayers of Vero cells in a six-well tissue culture plate.The cells were incubated at about 37° C. for 1 h, and the plates wereshaken every 15 min. The agarose overlay was then added. The overlay wasprepared by mixing equal volumes of a solution consisting of 100 ml2×MEM (Life Technologies) with sterile 2% agarose. Both solutions wereplaced in a 40° C. water bath for 1 h before adding the overlay. Thecells were incubated for 4 days at 37° C. in a humidified 5% CO₂-airmixture. A second overlay with an additional 4% neutral red was added onday 5. Virus plaques were counted about 12 h later.

Antigenicity of STF2Δ-Fusion Proteins

ELISA plates (96-well) were coated overnight at 4° C. with serialdilutions (100 μl/well) of purified STF2Δ-fusion proteins (SEQ ID NOS:761, 762, 657, 658, 673, 674) in PBS (about 2 m/ml). Plates were blockedwith 200 μl/well of Assay Diluent Buffer (ADB; BD Pharmingen) for onehour at room temperature. The plates were washed 3× in PBS-Tween, andthen incubated with antibodies reactive with flagellin or the E domainof the construct. The expression of flagellin was detected using the mAb6H11 (Intotek), while the antigenicity of WNV-E was monitored using apanel of mAb (5C5, 7H2, 5H10, 3A3, and 3D9) (Beasley, D. W., et al., J.Virol. 76:13097-13100 (2002)) were purchased from Bioreliance (RoadRockville, Md.). Antibodies diluted in ADB (about 100 μl/well) wereincubated overnight at 4° C. The plates were washed 3× with PBS-T.HRP-labeled goat anti-mouse IgG antibodies (Jackson Immunochemical, WestGrove, Pa.) diluted in ADB were added (100W/well) and the plates wereincubated at room temperature for 1 hour. The plates were washed 3× withPBS-T. After adding TMB (3,3′,5,5′-tetramentylbenzidine) Ultra substrate(Pierce Biotechnology, Rockford, Ill.) and monitoring color development,A₄₅₀ was measured on a Tecan Farcyte microspectrophotometer.

Immunization of Mice

C3H/HeN mice (10 per group) were immunized intraperitoneally orsubcutaneously with the indicated concentrations of fusion proteins orsynthetic peptides on days 0, 14 and 28. On days 21 and 35, immunizedanimals were bled by retro-orbital puncture. Sera were harvested byclotting and centrifugation of the heparin-free blood samples. On day35, mice were challenged with a lethal dose of WNV strain 2741 (Wang,T., et al., J. Immunol. 167:5273-5277 (2001)). Survival was monitoredfor 21 days post-challenge.

Serum Antibody Determination

West Nile envelope protein specific IgG levels were determined by ELISA.ELISA plates (96-wells) were coated overnight at about 4° C. with 100μl/well of West Nile E protein mAb 5C5, 7H2, 5H10, 3A3, and 3D9(Beasley, D. W., et al., J. Viro. 76:13097-13100 (2002)) (Bioreliance,Road Rockville, Md.) in PBS at a concentration of 2 μg/ml. Plates wereblocked with 200 μl/well of Assay Diluent Buffer (ADB; BD Pharmingen,San Diego Calif.) for one hour at room temperature. The plates werewashed 3× in PBS-T. Dilutions of the sera in ADB were added (100μl/well) and the plates were incubated overnight at 4° C. The plateswere washed 3× with PBS-T. HRP-labeled goat anti-mouse IgG antibodies(Jackson Immunochemical, West Grove, Pa.) diluted in ADB were added (100μl/well) and the plates were incubated at room temperature for 1 hour.The plates were washed 3× with PBS-T. After adding TMB(3,3′,5,5′-tetramentylbenzidine) Ultra substrate (Pierce Biotechnology,Rockford, Ill.) and monitoring color development, A₄₅₀ was measured on aTecan Farcyte microspectrophotometer.

Production of Pam3Cys.WNV001 Peptide Synthesis

Pam3Cys.WNV001 was synthesized by Bachem Bioscience, Inc. (King ofPrussia, Pa.). WNV001 is a 20 amino acid peptide (SEQ ID NO: 771) of theWest Nile virus envelope protein chemically coupled to atri-palmitoylcysteine (Pam3Cys) moiety through the amino terminal serineresidue of the peptide. The chemical name for Pam3Cys.WNV001 is[Palmitoyl-Cys((RS)-2,3-di(palmitoyloxy)-propyl)-LTSGHLKCRVKMEKLQLKGT(SEQ ID NO: 771) acetate salt]. The molecular mass of Pam3Cys.WNV001 is3163.3 daltons. The peptide was synthesized by Bachem using solid phasesynthesis methodologies and FMOC chemistry. The amino acid sequence ofPam3Cys.WNV001 was assembled on an H-Pro-2-chlorotrityl chloride resinby solid phase peptide synthesis. The peptide chain was elongated bysuccessive coupling of the amino acid derivatives. Each coupling stepwas preceded by an Fmoc-deprotection step and were accompanied byrepeated washing of the resin. After coupling of the last amino acidderivative, the final Fmoc-deprotection step was performed. Finally, thepeptide resin was washed and dried under reduced pressure. During solidphase peptide synthesis color indicator tests were performed for eachstep to monitor the completion of the Fmoc-cleavage and the subsequentcoupling of the amino acid derivatives. To couple Pam3Cys-OH to theelongated peptide, the lipid moiety was pre-activated withN,N′-dicyclohexyl-carbodiimide (DCCI) in the presence of1-hydroxybenzotriazole (HOBt). The resulting solution was filtered andadded to the peptide resin. At the end of the reaction time the peptideresin was washed and dried under reduced pressure. Color indicator testswere performed to control the coupling of Pam3Cys-OH. The completedpeptide was cleaved from the resin by incubating with trifluoroaceticacid (TFA). The liberated product (crude peptide material) wasprecipitated from the reaction mixture and lyophilized. The crudeproduct was used for initial immunogenicity studies.

Synthesis of WNV-E Peptide Arrays

Peptide arrays (SEQ ID NOS: 703-754) were synthesized by Sigma Genosys(Woodlands, Tex.).

Results

West Nile Fusion Protein

West Nile virus (WNV) has emerged in recent years in temperate regionsof Europe and North America, presenting a threat to public and animalhealth. The most serious manifestation of WNV infection is fatalencephalitis (inflammation of the brain) in humans and horses, as wellas mortality in certain domestic and wild birds. WNV has also been asignificant cause of human illness in the United States. The envelopeglycoprotein of West Nile (WNV-E) and other flaviviruses may generateneutralizing and protective antibodies. By linking this antigen to aToll-like Receptor ligand, the compositions, fusion proteins andpolypeptides described herein may target appropriate antigen presentingcells without the need for adjuvant or other immune modulatorformulations.

As described herein, several strategies have been implemented tofacilitate production of West Nile virus envelope (WNV-E) fusionproteins in E. coli. One approach is to engineer a smaller WNV-E antigenby fusing domain III (EIII) and, optionally, with amino acids of domainII of the WNV-E protein to full-length STF2 (e.g., STF2.E, STF2.EIII+).Domain III is responsible for virus-host interactions and retains manyWest Nile virus neutralizing antibody epitopes. It also contains only 2of the 12 cysteine residues present within the full length envelopeprotein, making expression in E. coli more feasible. A second approachhas been to delete the hyper-variable hinge region of flagellin (e.g.,STF2Δ) thereby creating a smaller fusion protein (STF2Δ.EIII+) Thehyper-variable region of flagellin is not required for TLR5 signalingand its removal may also reduce the immunogenic potential of flagellin.Both STF2.EIII+ and STF2Δ.EIII+ have been expressed in E. coli andpurified. The purified proteins have been characterized for TLR5signaling activity in bioassays and for E epitope display in ELISAassays using a panel of WNV-E polyclonal and neutralizing monoclonalantibodies. Results from these studies indicate that STF2Δ.EIII+ hashigher PAMP activity and more conformation-sensitive neutralizing WNV-Eepitopes than STF2.EIII+.

Purity of STF2.EIII+ and STF2Δ.EIII+

Several lots of STF2.EIII+ and STF2Δ.EIII+ have been produced in E. coliand purified (Table 12). STF2.EIII+ was expressed as a soluble proteinand purified under non-denaturing conditions using a 4-step process, asdescribed above, that included anion exchange chromatography and gelfiltration. Final yields from 6 L cultures ranged from about 0.9 mg toabout 3.8 mg and all preparations contained low levels of endotoxin asmeasured by standard LAL procedures (about <0.1 EU/μg protein, seesupra). In contrast, STF2Δ.EIII+ formed inclusion bodies in E. coli, andwas purified under denaturing conditions. All chromatography steps usedto purify STF2Δ.EIII+ required the use of 8M urea. Followingpurification, the denatured protein was refolded by step-wise dialysisto allow for gradual urea removal. Refolding was typically carried outat protein concentrations of about 0.3 mg/ml without any loss due toprotein precipitation. Two preparations of STF2Δ.EIII+ from a single 6 Lculture yielded about 1.2 and about 6.7 mg of protein, both of which hadacceptable endotoxin levels. As expected, purified STF2.EIII+ andSTF2Δ.EIII+ migrated on SDS PAGE under reducing conditions as about 65kDa and about 43 kDa proteins, respectively. Notably, STF2Δ.EIII+migrated slightly faster under non-reducing conditions. This alteredmigration may be due to disulfide bond formation involving the twocysteines residues in domain III of the envelope protein. As well, alarger species of STF2Δ.EIII+ was detected by Western blot analysiswhose molecular weight is consistent with a trimer form of the protein(“(STF2Δ.EIII+)×3 or 3 units of STF2Δ.EIII+”).

TABLE 11 Endotoxin levels and TLR-5 activity for STF2.EIII+ (SEQ ID NO:658) and STF2Δ.EIII+ (SEQ ID NO: 674) fusion proteins. Endotoxin BatchYield Levels Number Protein (mg) (EU/μg) TLR-5 EC₅₀ 052 STF2.EIII+ 3.80.03 >5000.00 ng/ml 054 STF2.EIII+ 0.9 0.02 1195.00 ng/ml 057 STF2.EIII+1.6 0.07 197.92 ng/ml 044 STF2Δ.EIII+ 1.2 0.07 1.13 ng/ml 045STF2Δ.EIII+ 6.7 0.07 4.34 ng/mlTLR5 Activity in the HEK293 IL-8 Assay

To compare the PAMP activity of both fusion proteins, a TLR5 bioassaywas performed. HEK293 IL-8 cells were treated with serial dilutions oftwo independent protein batches (FIGS. 62A and 62B). Cultures wereincubated for a 24 hour period and conditioned media were harvested andassayed for IL-8 production by ELISA. As shown in FIG. 62A, STF2Δ.EIII+showed potent TLR-5 activity. Regression analysis of the titration curvedetermined the EC₅₀ of batches 2004-044 and 2004-045 to be 1.13 ng/mland 4.34 ng/ml, respectively (Table 11, supra). In both cases, the TLR5specific-activity was at least about 10-fold higher than the controlprotein STF2.OVA. In contrast, 2 preparations of STF2.EIII+ showedsignificantly weaker TLR5 activity than STF2.OVA. The EC₅₀ of STF2.EIII+batches 054 and 057 were about 1195.00 ng/ml and about 197.92 ng/ml.

Antigenicity of STF2.EIII+ and STF2Δ.EIII+

The antigenicity of STF2.EIII+ and STF2Δ.EIII+ was examined by directELISA using a flagellin monoclonal antibody specific for the N-terminalregion of STF2 (6H11, Inotek Pharmaceuticals, Beverly, Mass.) and apanel of WNV-E-specific antibodies (5C5, 5H10, 3A3, 7H2 and 3D9,Bioreliance, Road Rockville, Md.) previously shown to neutralize WestNile virus in vitro. As shown in FIG. 63, a comparison of the reactivityof full length West Nile virus envelope protein with STF2Δ.EIII+revealed that West Nile virus monoclonal antibodies 5C5, 5H10, 3A3 and7H2, but not 3D9 recognize the fusion protein. This pattern ofreactivity is consistent with the proposed location of 5C5, 5H10, 3A3and 7H2 epitopes within EIII. The epitope for 3D9 lies outside of domainIII of the West Nile virus envelope protein. As expected, all West Nilevirus monoclonal antibodies reacted with full length West Nile virusenvelope protein and the flagellin monoclonal only reacted withSTF2Δ.EIII+. Both proteins reacted with a polyclonal West Nile virusenvelope antiserum, but STF2Δ.EIII+ reactivity was somewhat reduced,perhaps due to the reduced number of potential epitopes present in thesmaller domain.

Using 5C5 and 7H10 WNV monoclonal antibodies, a direct antigeniccomparison was made between STF2.EIII+ and STF2Δ.EIII+ (FIGS. 64A, 64B,64C and 64D). In these studies, plates were coated with the indicatedproteins and then detected with polyclonal rabbit anti-E, or mousemonoclonal antibodies as described. As shown in FIGS. 64A, 64B, 64C and64D, both STF2.EIII+ and STF2Δ.EIII+ were readily detected with theflagellin monoclonal antibody with no significant differences inreactivity. However, distinct reactivity with the anti-envelopemonoclonal antibodies was observed. The reactivity of STF2Δ.EIII+ witheither 5C5 or 7H2 was significantly greater than that observed withSTF2.EIII+. Collectively, these results indicate that the flagellin 6H11epitope of STF2Δ.EIII+ is uncompromised and is comparable to theflagellin sequence of STF2.EIII+. They also highlight distinctdifferences in the antigenicity of the EIII domains of these proteinsand indicate that STF2Δ.EIII+ contains more of the critical conformationdependent neutralizing epitopes than STF2.EIII+.

Efficacy and Immunogenicity

Several efficacy studies designed to examine the protective efficacy ourcandidates in C3H/HeN mice following challenge with West Nile virus havebeen completed. Studies typically consisted of 5 groups of mice (10 miceper group) immunized intraperitoneally (i.p.) or subcutaneously (s.c.)on days 0, 14 and 28. On days 21 and 35, sera were harvested and testedfor West Nile virus envelope protein-IgG antibody (ELISA) and theability to neutralize West Nile virus in vitro (PRNT assay). On day 35,mice were challenged with a lethal dose of West Nile virus strain 2741.Survival was monitored for 21 days post-challenge.

Mice were immunized with PBS, Drosophila conditioned medium containingSTF2.E (CM, positive control), 25 μg of STF2Δ.EIII+ i.p., 25 μgSTF2Δ.EIII+s.c., 25 μg STF2.EIII+ i.p. and 25 μg STF2.EIII+ s.c. TheWest Nile virus envelope protein antibody responses and survival dataare shown FIGS. 65 and 66. By day 35 all groups that receivedSTF2Δ.EIII+ had significant levels of West Nile virus envelope proteinIgG. In contrast, mice that received STF2.EIII+ had no measurable WestNile virus envelope protein antibody response. Administration ofSTF2Δ.EIII+ i.p. or s.c led to 100% survival following West Nile viruschallenge. Consistent with the poor immunogenicity of STF2.EIII+, littleto no protection was provided by this candidate when compared to the PBScontrol. The poor immunogenicity and efficacy of STF2.EIII+ in thisstudy are attributed to the reduced TLR5 activity and/or the weak EIIIepitope reactivity of this protein.

Plaque Reduction Neutralization Titers

To further evaluate the West Nile virus envelope protein antibodyresponse elicited by STF2Δ.EIII+ and potentially correlate protectiveefficacy with neutralizing antibody titers, the plaque reductionneutralization test (PRNT) was performed. Day 35 serum samples fromefficacy studies described above were tested for their ability to blockWest Nile virus infection in cultured Vero cells. Briefly, pooled mouseserum samples were heat-inactivated and serially diluted two-fold in PBSwith 0.5% gelatin. Dilutions starting with 1:10 were incubated withabout 100 pfu of the West Nile virus strain 2741. The virus/serummixture was incubated at about 37° C. for 1 h and then inoculated ontoconfluent monolayers of Vero cells (ATCC, Catalog Number CCL-81,Manassas, Va.) in duplicate wells of 6-well tissue culture plates. Thevirus was allowed to adsorb to the cell monolayer prior to adding a 1%agarose overlay. Infected cell cultures were incubated for 4 days at 37°C. followed by a second agarose overlay containing 4% neutral red. Virusplaques were counted 12 h later. Serum titers that led to 80% reductionin viral plaque numbers (PRNT₈₀) were recorded.

A summary of the PRNT₈₀ data from efficacy studies concerning STF2.EIII+and STF2Δ.EIII+ is presented in Table 12 below. In two independentstudies involving STF2.EIII+ where survival of about 50% or less wasreported, pooled sera failed to inhibit plaque formation. This findingis not surprising given the weak antibody response elicited by thisconstruct. In three efficacy studies involving STF2Δ.EIII+ wheresurvival was about 70% or greater, pooled sera had neutralization titersof 1:40 or better. Neutralization titers of 1:40 or greater typicallycorrelate with protection in vivo.

TABLE 12 Survivial and PRNT₈₀ Results for STF2.EIII+ (SEQ ID NO: 658),STF2Δ.EIII+ (SEQ ID NO: 674) and STF2.E (SEQ ID NO: 762) CM (ControlMedia) Fusion Proteins Batch Candidate Route Study # Survival (%) PRNT₈₀(dilution) 054 STF2.EIII+ i.p. 3 50 Negative 057 STF2.EIII+ i.p. 4 11Negative 057 STF2.EIII+ s.c. 4 20 negative 044 STF2Δ.EIII+ i.p. 2 701:40 045 STF2Δ.EIII+ i.p. 3 90 1:40 045 STF2Δ.EIII+ s.c. 3 100  1:160045 STF2Δ.EIII+ i.p. 4 100 1:80 045 STF2Δ.EIII+ s.c. 4 100 1:40 — STF2.ECM i.p. 3 90  1:640 — STF2.E CM i.p. 4 —  1:1280STF2Δ.EIIIs+ a Modified Version of STF2Δ.EIII+

Protein preparations of STF2Δ.EIII+ tested in the mouse efficacy studiesdescribed above were purified by anion-exchange and size-exclusionchromatography steps carried out under denaturing conditions followed byrefolding using step-wise dialysis. With this process, two predominantspecies that correspond to the monomeric and trimeric forms ofSTF2Δ.EIII+ were generated and present as a mixture in the finalproduct. To minimize the heterogeneity of the final product, newrefolding and purification methods have been developed that favor theproduction of either monomer or trimer. Because it is unclear which formof STF2Δ.EIII+ is the active component or if both are equally potent,both species have been produced in milligram quantities and tested forefficacy in mice.

It was initially unclear as to why STF2Δ.EIII+ refolding resulted in theformation of a trimeric species. However, when the sequence of theSTF2Δ.EIII+ expression construct was re-examined, we identified acysteine residue within the linker sequence that separates STF2Δ, fromEIII+. The presence of this cysteine would likely interfere with theformation of the appropriate disulfide bond during refolding and mightaccount for the trimeric form of STF2Δ.EIII+. This unnecessary cysteinewas changed to a serine using site-directed mutagenesis and the modifiedprotein (STF2Δ.EIIIs+) was produced and purified. It should be notedthat refolding the serine-substituted construct yielded only monomericprotein.

Protective efficacy of STF2Δ.EIII+ (monomer) and STF2Δ.EIIIs+ (trimer)were evaluated in C3H/HeN mice following challenge with West Nile virus.Five groups of mice (10 per group) were immunized with about 25 ug ofprotein s.c. on days 0, 14 and 28. On days 21 and 35, sera wereharvested and tested for WNV-E IgG antibody (ELISA). On day 38, micewere challenged with a lethal dose of WNV strain 2741 and survival wasmonitored for 21 days. ELISA results from boost 2 (day 35, FIG. 67) andsurvival data (FIG. 68) indicate that all constructs elicitedsignificant levels of WNV-E reactive IgG prior to viral challenge andprovided about 90% to about 100% protection against the lethalinfection. These findings indicate that monomeric or multimeric (e.g.,trimers) forms of STFΔ.EIII+ are efficacious and removal of theadditional cysteine from the construct does not appreciably impactpotency. Removal of the cysteine within the linker sequence may simplifypurification of the protein by reducing heterogeneity following proteinrefolding.

CONCLUSION

Two recombinant fusion proteins containing the Salmonella typhimuriumflagellin (STF2) fused to EIII+ domain of West Nile virus envelopeprotein have been generated. One includes the full length STF2 sequence(STF2.EIII+) and the other a modified version of STF2 that lacks theinternal hypervariable region of STF2 (STF2Δ.EIII+). Both proteins havebeen expressed in E. coli and purified by conventional means using anionexchange chromatography and gel filtration. STF2.EIII+ was produced as asoluble protein and was purified under non-denaturing conditions. Incontrast, STF2Δ.EIII+ was expressed as an insoluble protein and waspurified under denaturing conditions and refolded by step-wise dialysisto remove urea. In HEK293 IL8 assays, preparations of STF2Δ.EIII+ showedgreater TLR-5 activity than STF2.EIII+.

In envelope protein epitope display analysis using ELISA assays and WestNile virus envelope protein antibodies, STF2Δ.EIII+ displayed more ofthe critical conformation dependent neutralizing epitopes. Consistentwith the potent TLR-5 activity and envelope protein epitope antigenicityobserved with STF2Δ.EIII+, STF2Δ.EIII+ was highly immunogenic andefficacious in mice challenged with a lethal dose of West Nile virus.Because monomeric and trimeric species of STF2Δ.EIII+ were generatedduring the purification process of this protein, a cysteine within thelinker sequence of the expression construct was changed to a serine.Removal of this cysteine eliminated the production of trimeric forms ofthe protein during refolding and resulted in the generation of monomericproduct that displayed potent efficacy in vivo.

Japanese Encephalitis Fusion Protein

JE virus is localized in Asia and northern Australia (about 50,000 caseswith about 10,000 deaths annually). An approved inactivated virusvaccine was recently associated with a case of acute disseminatedencephalomyelitis, prompting the Japanese Ministry of Health, Labor andWelfare to recommend the nationwide suspension of the vaccine. Given thecomplexities of producing inactivated viruses in infected mouse brainsor even in cell culture, and the potential for adverse events associatedwith inactivated viruses, the opportunity for recombinant-based JEvaccine is appealing.

A STF2Δ.JEIII+ fusion construct was constructed. The JE EIII+ DNAfragment was generated synthetically and codon optimized for expressionin E. coli. The sequence was ligated into pET24STF2Δ to generatepETSTF2Δ.JEIII+. Expression constructs have been screened by restrictionanalysis and for expression in E. coli BLR(DE3) by IPTG induction. TheDNA sequence of each construct has been confirmed, and production of theprotein has been scaled up. A batch of material has been generated. Atotal of about 24 mg of material was purified. This material has potentTLR5 activity, acceptable levels of endotoxin (about 0.03 EU/μg) and aA280/A260 ratio of about 1.3.

Flavivirus Peptides

Identification of WNV-E Specific Antibody Epitopes

To identify linear epitopes within the West Nile virus envelope proteinthat are recognized by antisera from STFΔ.EIIIs+ immunized mice, severalsynthetic peptide arrays were generated. One array consisted ofoverlapping peptides of 20 amino acids in length that spanned the entireWest Nile virus domain III and parts of domain II (SEQ ID NOS: 728-754).ELISA results with this array identified a highly reactive 20 amino acidsequence that mapped to the N-terminal region of domain III and includedpart of the domain I domain CRVKMEKLQLKGTTYGVCSK (SEQ ID NO: 728). Tofine map this epitope, additional arrays were generated that focused onthe domain I and II junctions (SEQ ID NOS: 703-754). These arraysincluded an alanine substitution scan to identify amino acids criticalfor antibody binding (SEQ ID NOS: 728-754). As shown in FIGS. 69 and 70,antisera from STF2Δ.EIII (monomer and trimer) and STF2Δ.EIIIs+ immunizedmice reacted with peptides that spanned the EI/EIII junction (peptidesE-30 to E-42) and included the E2-21 peptide CRVKMEKLQLKGTTYGVCSK (SEQID NO: 728). This reactivity was severely reduced when specific aminoacids (E6, K7, L10 and K11) were changed to alanines (FIG. 71). Althoughit is not known if the antibodies that recognize this epitope areneutralizing, efforts are underway to design and test a peptide vaccinebased on this region of WNV-E.

Immunogenicity of Pam3Cys.WNV001 Peptide Vaccine

A lipidated West Nile virus envelope protein fused to Pam3Cys on theN-terminal end was synthesized using the 20 amino acid sequenceLTSGHLKCRVKMEKLQLKGT (SEQ ID NO:772) (Putnak, R., et al, Vaccine23:4442-4452 (2005)). The immunogenicity of this peptide was tested inC3H/HeN mice and compared to peptide without Pam3Cys (FIG. 72). Thereactivity of antisera from immunized animals was tested by direct ELISAas described in the legend and the results indicate that thePam3Cys.WNV001 peptide is significantly more immunogenic than thepeptide without the TLR2 modification. The antisera from these studieswill be tested in virus neutralization assays (PRNT) to determine if theantibodies elicited will neutralize West Nile virus in vitro. Thelipidated peptide will also be tested in the West Nile virus challengemodel to assess protective efficacy against a lethal virus challenge.

Assay Development

Competition ELISA Assay Development

To assess the neutralizing potential of antisera derived from immunizedmice, a competition ELISA assay was developed using well-characterizedmonoclonal antibody (7H2) that neutralizes West Nile virus in cultureand reacts with a conformation-sensitive epitope within the EIII domainof the West Nile virus envelope protein antigen. The assay was designedas a capture ELISA that measures the ability of sera from immunizedanimals to prevent 7H2 from binding West Nile virus envelope protein.Serial dilutions ranging from 1:10 to 1:5000 of day 35 mouse antiserafrom efficacy study 4 (FIGS. 65 and 66, Table 13) were incubated withbiotinylated West Nile virus envelope protein and then added to ELISAplates pre-coated with 7H2 monoclonal antibody (Bioreliance, RoadRockville, Md.). Following several washes to remove unbound material,bound West Nile virus envelope protein was detected using avidin-HRP.Results from a representative experiment are shown in FIG. 69. Atdilutions of 1:25, a measurable loss of West Nile virus envelope proteinbinding to 7H2-coated plates was observed when antisera derived fromanimals immunized with STF2Δ.EIIIs where tested. No competition wasdetected with antisera derived from mock immunized animals that receivedPBS in place of antigen. These initial results demonstrate thatantibodies elicited by STF2Δ.EIII+ compete with 7H2 for binding WestsNile virus envelope protein. These findings are consistent with theprotection from WNV infection observed in animals immunized withSTF2Δ.EIII+ and help establish a correlation between antibody epitopereactivity in vitro and efficacy in vivo.

While this invention has been particularly shown and described withreferences to example embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

What is claimed is:
 1. A method of making an immunogenic fusion protein,comprising the steps of: a) transforming a nucleic acid encoding afusion protein into a prokaryotic host cell, the fusion proteinincluding, i) at least a portion of a flagellin that initiates anintracellular signal transduction pathway for a Toll-like Receptor, andii) a portion of an influenza viral hemagglutinin, wherein the portionof the influenza viral hemagglutinin lacks a transmembrane domain, acytoplasmic domain; and an HA2 subunit, and includes, at least a portionof a globular head; and at least a portion of at least one secondarystructure having at least one β-sheet at a bottom of the globular headthat causes the globular head to essentially retain its tertiarystructure; and b) culturing the prokaryotic host cell to thereby makethe immunogenic fusion protein.
 2. The method of claim 1, wherein theportion of the globular head includes a substrate binding domain, atleast two disulfide bonds and at least two alpha helices.
 3. The methodof claim 2, wherein the fusion protein stimulates a protective immuneresponse in a subject.
 4. The method of claim 1, wherein the portion ofthe influenza viral hemagglutinin further includes at least oneβ-sandwich at the bottom of the portion of the globular head.
 5. Themethod of claim 4, wherein the portion of the influenza viralhemagglutinin further includes at least two β-strands at the bottom ofthe portion of the globular head.
 6. The method of claim 1, wherein theportion of the influenza viral hemagglutinin includes an HA1-1 portionof the influenza viral hemagglutinin.
 7. The method of claim 1, whereinthe portion of the influenza viral hemagglutinin includes an HA1-2portion of the influenza viral hemagglutinin.
 8. The method of claim 1,wherein the nucleic acid encoding the flagellin lacks a nucleotidesequence encoding at least a portion of the hinge region of theflagellin.
 9. The method of claim 1, wherein the flagellin includes atleast one member selected from the group consisting of Salmonellatyphimurium flagellin, an E. coli flagellin, a S. muenchen flagellin, aYersinia flagellin, a P. aeruginosa flagellin and a L. monocytogenesflagellin.
 10. The method of claim 1, further including the step ofoperably linking a nucleotide sequence encoding an amino acid linkerbetween a nucleotide sequence encoding the flagellin and a nucleotidesequence encoding the influenza viral hemagglutinin to thereby form thenucleic acid encoding the fusion protein.
 11. The method of claim 1,wherein the influenza viral hemagglutinin protein is an influenza Aviral hemagglutinin protein.
 12. The method of claim 11, wherein theinfluenza A is at least one member selected from the group consisting ofH1, H2, H3, H5, H7 and H9.
 13. The method of claim 1, wherein theinfluenza viral hemagglutinin protein is an influenza B viralhemagglutinin protein.
 14. The method of claim 1, wherein the influenzaviral hemagglutinin protein is an influenza C viral hemagglutininprotein.
 15. The method of claim 1, wherein the prokaryotic host cell isan E. coli prokaryotic host cell.
 16. The method of claim 1, wherein theprokaryotic host cell is at least one member selected from the groupconsisting of a Pseudomonas prokaryotic host cell, a Bacillusprokaryotic host cell and a Salmonella prokaryotic host cell.
 17. Themethod of claim 1, further including the step of substituting at leastone nucleotide sequence encoding at least one amino acid residueselected from the group consisting of a hydrophilic amino acid residue,a polar amino acid residue and a neutral amino acid residue, for atleast one nucleotide sequence that encodes at least one correspondinghydrophobic amino acid residue in the portion of the influenza viralhemagglutinin.
 18. The method of claim 17, wherein the hydrophobic aminoacid residue includes at least one member selected from the groupconsisting of a phenylalanine residue, a tryptophan residue and atyrosine residue.
 19. The method of claim 17, wherein the polar aminoacid residue includes at least one member selected from the groupconsisting of an aspartic acid residue and a glutamic acid residue. 20.The method of claim 1, wherein the portion of the influenza viralhemagglutinin further lacks a signal sequence.
 21. The method of claim1, wherein the portion of the influenza viral hemagglutinin furtherincludes at least one alpha helix.
 22. The method of claim 1, whereinthe portion of the influenza viral hemagglutinin further includes atleast one member selected from the group consisting of a salt bridge, aleucine zipper and a zinc finger.
 23. The method of claim 1, wherein theportion of the influenza viral hemagglutinin further includes at leastone member selected from the group consisting of at least one disulfidebond, at least two disulfide bonds, at least four disulfide bonds, atleast five disulfide bonds and at least six disulfide bonds.
 24. Themethod of claim 1, further including the step of transforming theprokaryotic host cell with a chaperone nucleic acid sequence.
 25. Themethod of claim 24, wherein the chaperone nucleic acid sequence is atleast one member selected from the group consisting of a groES-groELchaperone, a dnaK-dnaJ-grpE chaperone, a groES-groEL-tig chaperone and atig chaperone.
 26. The method of claim 1, further including the step ofoperably linking a nucleotide sequence encoding a carrier protein to anucleotide sequence encoding the flagellin or to a nucleotide sequenceencoding the portion of the influenza viral hemagglutinin to therebyform the nucleic acid encoding the fusion protein.
 27. The method ofclaim 26, wherein the carrier protein is at least one member selectedfrom the group consisting of tetanus toxoid, a Vibrio cholerae toxoid, adiphtheria toxoid, a cross-reactive mutant of diphtheria toxoid, a E.coli B subunit of a heat labile enterotoxin, a tobacco mosaic virus coatprotein, a rabies virus envelope protein, a rabies virus envelopeglycoprotein, a thyroglobulin, a heat shock protein 60, a keyhole limpethemocyanin and an early secreted antigen tuberculosis-6.
 28. The methodof claim 1, further including the step of fusing at least a portion of acarrier protein to the fusion protein.
 29. A method of making animmunogenic fusion protein, comprising the steps of: a) transforming anucleic acid encoding a fusion protein into a eukaryotic host cell,wherein the eukaryotic host cell is not a Pichia pastoris eukaryotichost cell, the fusion protein including, i) at least a portion of aflagellin that initiates an intracellular signal transduction pathwayfor a Toll-like Receptor, and ii) a portion of an influenza viralhemagglutinin, wherein the portion of the influenza viral hemagglutininlacks a transmembrane domain, a cytoplasmic domain; and an HA2 subunit,and includes, at least a portion of a globular head, and at least aportion of at least one secondary structure having at least one β-sheetat a bottom of the globular head that causes the globular head toessentially retain its tertiary structure; and b) culturing theeukaryotic host cell to thereby make the immunogenic fusion protein. 30.The method of claim 29, wherein the portion of the globular headincludes a substrate binding domain, at least two disulfide bonds and atleast two alpha helices.
 31. The method of claim 30, wherein the fusionprotein stimulates a protective immune response in a subject.
 32. Themethod of claim 29, wherein the portion of the influenza viralhemagglutinin further includes at least one β-sandwich at the bottom ofthe portion of the globular head.
 33. The method of claim 32, whereinthe portion of the influenza viral hemagglutinin further includes atleast two β-strands at the bottom of the portion of the globular head.34. The method of claim 29, wherein the portion of the influenza viralhemagglutinin includes an HA1-1 portion of the influenza viralhemagglutinin.
 35. The method of claim 29, wherein the portion of theinfluenza viral hemagglutinin includes an HA1-2 portion of the influenzaviral hemagglutinin.
 36. The method of claim 29, wherein the nucleicacid encoding the flagellin lacks a nucleotide sequence encoding atleast a portion of the hinge region of the flagellin.
 37. The method ofclaim 29, wherein the flagellin includes at least one member selectedfrom the group consisting of Salmonella typhimurium flagellin, an E.coli flagellin, a S. muenchen flagellin, a Yersinia flagellin, a P.aeruginosa flagellin and a L. monocytogenes flagellin.
 38. The method ofclaim 29, further including the step of operably linking a nucleotidesequence encoding an amino acid linker between a nucleotide sequenceencoding the flagellin and a nucleotide sequence encoding the influenzaviral hemagglutinin to thereby form the nucleic acid encoding the fusionprotein.
 39. The method of claim 29, wherein the influenza viralhemagglutinin protein is an influenza A viral hemagglutinin protein. 40.The method of claim 39, wherein the influenza A is at least one memberselected from the group consisting of H1, H2, H3, H5, H7 and H9.
 41. Themethod of claim 19, wherein the influenza viral hemagglutinin protein isan influenza B viral hemagglutinin protein.
 42. The method of claim 29,wherein the influenza viral hemagglutinin protein is an influenza Cviral hemagglutinin protein.
 43. The method of claim 29, wherein theeukaryotic host cell is a yeast host cell.
 44. The method of claim 29,wherein the eukaryotic host cell is at least one member selected fromthe group consisting of a fungal eukaryotic host cell, a parasiteeukaryotic host cell and an insect eukaryotic host cell.
 45. The methodof claim 44, wherein the insect eukaryotic host cell is a baculovirusinsect eukaryotic host cell.
 46. The method of claim 44, wherein theinsect eukaryotic host cell is a Drosophila insect eukaryotic host cell.47. The method of claim 29, wherein the eukaryotic host cell is aChinese hamster ovary eukaryotic host cell.
 48. The method of claim 29,wherein the eukaryotic host cell is a Saccharomyces eukaryotic hostcell.
 49. The method of claim 29, further including the step ofsubstituting at least one nucleotide sequence encoding at least oneamino acid residue selected from the group consisting of a hydrophilicamino acid residue, a polar amino acid residue and a neutral amino acidresidue, for at least one nucleotide sequence that encodes at least onecorresponding hydrophobic amino acid residue in the portion of theinfluenza viral hemagglutinin.
 50. The method of claim 49, wherein thehydrophobic amino acid residue includes at least one member selectedfrom the group consisting of a phenylalanine residue, a tryptophanresidue and a tyrosine residue.
 51. The method of claim 50, wherein thepolar amino acid residue includes at least one member selected from thegroup consisting of an aspartic acid residue and a glutamic acidresidue.
 52. The method of claim 29, wherein the portion of theinfluenza viral hemagglutinin further lacks a signal sequence.
 53. Themethod of claim 29, wherein the portion of the influenza viralhemagglutinin further includes at least one alpha helix.
 54. The methodof claim 29, wherein the portion of the influenza viral hemagglutininfurther includes at least one member selected from the group consistingof a salt bridge, a leucine zipper and a zinc finger.
 55. The method ofclaim 29, wherein the portion of the influenza viral hemagglutininfurther includes at least one member selected from the group consistingof at least one disulfide bond, at least two disulfide bonds, at leastfour disulfide bonds, at least five disulfide bonds and at least sixdisulfide bonds.
 56. The method of claim 29, further including the stepof transforming the prokaryotic host cell with a chaperone nucleic acidsequence.
 57. The method of claim 56, wherein the chaperone nucleic acidsequence is at least one member selected from the group consisting of agroES-groEL chaperone, a dnaK-dnaJ-grpE chaperone, a groES-groEL-tigchaperone and a tig chaperone.
 58. The method of claim 29, furtherincluding the step of operably linking a nucleotide sequence encoding acarrier protein to a nucleotide sequence encoding the flagellin or to anucleotide sequence encoding the portion of the influenza viralhemagglutinin to thereby form the nucleic acid encoding the fusionprotein.
 59. The method of claim 28, wherein the carrier protein is atleast one member selected from the group consisting of tetanus toxoid, aVibrio cholerae toxoid, a diphtheria toxoid, a cross-reactive mutant ofdiphtheria toxoid, a E. coli B subunit of a heat labile enterotoxin, atobacco mosaic virus coat protein, a rabies virus envelope protein, arabies virus envelope glycoprotein, a thyroglobulin, a heat shockprotein 60, a keyhole limpet hemocyanin and an early secreted antigentuberculosis-6.
 60. The method of claim 29, further including the stepof fusing at least a portion of a carrier protein to the fusion protein.